Publications by authors named "Anna V Golubeva"

25 Publications

  • Page 1 of 1

Bifidobacterium longum counters the effects of obesity: Partial successful translation from rodent to human.

EBioMedicine 2021 Jan 18;63:103176. Epub 2020 Dec 18.

APC Microbiome Ireland, University College Cork, Cork, Ireland; Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland.

Background: The human gut microbiota has emerged as a key factor in the development of obesity. Certain probiotic strains have shown anti-obesity effects. The objective of this study was to investigate whether Bifidobacterium longum APC1472 has anti-obesity effects in high-fat diet (HFD)-induced obese mice and whether B. longum APC1472 supplementation reduces body-mass index (BMI) in healthy overweight/obese individuals as the primary outcome. B. longum APC1472 effects on waist-to-hip ratio (W/H ratio) and on obesity-associated plasma biomarkers were analysed as secondary outcomes.

Methods: B. longum APC1472 was administered to HFD-fed C57BL/6 mice in drinking water for 16 weeks. In the human intervention trial, participants received B. longum APC1472 or placebo supplementation for 12 weeks, during which primary and secondary outcomes were measured at the beginning and end of the intervention.

Findings: B. longum APC1472 supplementation was associated with decreased bodyweight, fat depots accumulation and increased glucose tolerance in HFD-fed mice. While, in healthy overweight/obese adults, the supplementation of B. longum APC1472 strain did not change primary outcomes of BMI (0.03, 95% CI [-0.4, 0.3]) or W/H ratio (0.003, 95% CI [-0.01, 0.01]), a positive effect on the secondary outcome of fasting blood glucose levels was found (-0.299, 95% CI [-0.44, -0.09]).

Interpretation: This study shows a positive translational effect of B. longum APC1472 on fasting blood glucose from a preclinical mouse model of obesity to a human intervention study in otherwise healthy overweight and obese individuals. This highlights the promising potential of B. longum APC1472 to be developed as a valuable supplement in reducing specific markers of obesity.

Funding: This research was funded in part by Science Foundation Ireland in the form of a Research Centre grant (SFI/12/RC/2273) to APC Microbiome Ireland and by a research grant from Cremo S.A.
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http://dx.doi.org/10.1016/j.ebiom.2020.103176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7838052PMC
January 2021

Strain differences in behaviour and immunity in aged mice: Relevance to Autism.

Behav Brain Res 2021 Feb 20;399:113020. Epub 2020 Nov 20.

APC Microbiome Ireland, University College Cork, Ireland; Department of Anatomy and Neuroscience, University College Cork, Ireland. Electronic address:

The BTBR mouse model has been shown to be associated with deficits in social interaction and a pronounced engagement in repetitive behaviours. Autism spectrum disorder (ASD) is the most prevalent neurodevelopmental condition globally. Despite its ubiquity, most research into the disorder remains focused on childhood, with studies in adulthood and old age relatively rare. To this end, we explored the differences in behaviour and immune function in an aged BTBR T + Itpr3tf/J mouse model of the disease compared to a similarly aged C57bl/6 control. We show that while many of the alterations in behaviour that are observed in young animals are maintained (repetitive behaviours, antidepressant-sensitive behaviours, social deficits & cognition) there are more nuanced effects in terms of anxiety in older animals of the BTBR strain compared to C57bl/6 controls. Furthermore, BTBR animals also exhibit an activated T-cell system. As such, these results represent confirmation that ASD-associated behavioural deficits are maintained in ageing, and that that there may be need for differential interventional approaches to counter these impairments, potentially through targeting the immune system.
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http://dx.doi.org/10.1016/j.bbr.2020.113020DOI Listing
February 2021

Enduring Behavioral Effects Induced by Birth by Caesarean Section in the Mouse.

Curr Biol 2020 Oct 20;30(19):3761-3774.e6. Epub 2020 Aug 20.

APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland; Department of Anatomy and Neuroscience, University College Cork, Cork T12 XF62, Ireland. Electronic address:

Birth by Caesarean (C)-section impacts early gut microbiota colonization and is associated with an increased risk of developing immune and metabolic disorders. Moreover, alterations of the microbiome have been shown to affect neurodevelopmental trajectories. However, the long-term effects of C-section on neurobehavioral processes remain unknown. Here, we demonstrated that birth by C-section results in marked but transient changes in microbiome composition in the mouse, in particular, the abundance of Bifidobacterium spp. was depleted in early life. Mice born by C-section had enduring social, cognitive, and anxiety deficits in early life and adulthood. Interestingly, we found that these specific behavioral alterations induced by the mode of birth were also partially corrected by co-housing with vaginally born mice. Finally, we showed that supplementation from birth with a Bifidobacterium breve strain, or with a dietary prebiotic mixture that stimulates the growth of bifidobacteria, reverses selective behavioral alterations in C-section mice. Taken together, our data link the gut microbiota to behavioral alterations in C-section-born mice and suggest the possibility of developing adjunctive microbiota-targeted therapies that may help to avert long-term negative consequences on behavior associated with C-section birth mode.
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http://dx.doi.org/10.1016/j.cub.2020.07.044DOI Listing
October 2020

Naturally Derived Polyphenols Protect Against Corticosterone-Induced Changes in Primary Cortical Neurons.

Int J Neuropsychopharmacol 2019 12;22(12):765-777

APC Microbiome Ireland.

Background: Polyphenols are phytochemicals that have been associated with therapeutic effects in stress-related disorders. Indeed, studies suggest that polyphenols exert significant neuroprotection against multiple neuronal injuries, including oxidative stress and neuroinflammation, but the mechanisms are unclear. Evidence indicates that polyphenol neuroprotection may be mediated by activation of Nrf2, a transcription factor associated with antioxidant and cell survival responses. On the other hand, in stress-linked disorders, Fkbp5 is a novel molecular target for treatment because of its capacity to regulate glucocorticoid receptor sensitivity. However, it is not clear the role Fkbp5 plays in polyphenol-mediated stress modulation. In this study, the neuroprotective effects and mechanisms of the naturally derived polyphenols xanthohumol and quercetin against cytotoxicity induced by corticosterone were investigated in primary cortical cells.

Methods: Primary cortical cells containing both neurons and astrocytes were pre-incubated with different concentrations of quercetin and xanthohumol to examine the neuroprotective effects of polyphenols on cell viability, morphology, and gene expression following corticosterone insult.

Results: Both polyphenols tested prevented the reduction of cell viability and alterations of neuronal/astrocytic numbers due to corticosterone exposure. Basal levels of Bdnf mRNA were also decreased after corticosterone insult; however, this was reversed by both polyphenol treatments. Interestingly, the Nrf2 inhibitor blocked xanthohumol but not quercetin-mediated neuroprotection. In contrast, we found that Fkbp5 expression is exclusively modulated by quercetin.

Conclusions: These results suggest that naturally derived polyphenols protect cortical cells against corticosterone-induced cytotoxicity and enhance cell survival via modulation of the Nrf2 pathway and expression of Fkbp5.
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http://dx.doi.org/10.1093/ijnp/pyz052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929673PMC
December 2019

Short-chain fatty acids and microbiota metabolites attenuate ghrelin receptor signaling.

FASEB J 2019 12 9;33(12):13546-13559. Epub 2019 Oct 9.

Alimentary Pharmabiotic Centre (APC) Microbiome Ireland, University College Cork, Cork, Ireland.

The gastrointestinal microbiota is emerging as a unique and inexhaustible source for metabolites with potential to modulate G-protein coupled receptors (GPCRs). The ghrelin receptor [growth hormone secretagogue receptor (GHSR)-1a] is a GPCR expressed throughout both the gut and the brain and plays a crucial role in maintaining energy balance, metabolism, and the central modulation of food intake, motivation, reward, and mood. To date, few studies have investigated the potential of the gastrointestinal microbiota and its metabolites to modulate GPCR signaling. Here we investigate the ability of short-chain fatty acids (SCFAs), lactate, and different bacterial strains, including and genera, to modulate GHSR-1a signaling. We identify, for what is to our knowledge the first time, a potent effect of microbiota-derived metabolites on GHSR-1a signaling with potential significant consequences for host metabolism and physiology. We show that SCFAs, lactate, and bacterial supernatants are able to attenuate ghrelin-mediated signaling through the GHSR-1a. We suggest a novel route of communication between the gut microbiota and the host modulation of GHSR-1a receptor signaling. Together, this highlights the emerging therapeutic potential in the exploration of the microbiota metabolome in the specific targeting of key GPCRs, with pleiotropic actions that span both the CNS and periphery.-Torres-Fuentes, C., Golubeva, A. V., Zhdanov, A. V., Wallace, S., Arboleya, S., Papkovsky, D. B., El Aidy, S., Ross, P., Roy, B. L., Stanton, C., Dinan, T. G., Cryan, J. F., Schellekens, H. Short-chain fatty acids and microbiota metabolites attenuate ghrelin receptor signaling.
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http://dx.doi.org/10.1096/fj.201901433RDOI Listing
December 2019

The Microbiota-Gut-Brain Axis.

Physiol Rev 2019 10;99(4):1877-2013

APC Microbiome Ireland, University College Cork, Cork, Ireland; Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland; Department of Psychiatry and Neurobehavioural Science, University College Cork, Cork, Ireland; and Department of Physiology, University College Cork, Cork, Ireland.

The importance of the gut-brain axis in maintaining homeostasis has long been appreciated. However, the past 15 yr have seen the emergence of the microbiota (the trillions of microorganisms within and on our bodies) as one of the key regulators of gut-brain function and has led to the appreciation of the importance of a distinct microbiota-gut-brain axis. This axis is gaining ever more traction in fields investigating the biological and physiological basis of psychiatric, neurodevelopmental, age-related, and neurodegenerative disorders. The microbiota and the brain communicate with each other via various routes including the immune system, tryptophan metabolism, the vagus nerve and the enteric nervous system, involving microbial metabolites such as short-chain fatty acids, branched chain amino acids, and peptidoglycans. Many factors can influence microbiota composition in early life, including infection, mode of birth delivery, use of antibiotic medications, the nature of nutritional provision, environmental stressors, and host genetics. At the other extreme of life, microbial diversity diminishes with aging. Stress, in particular, can significantly impact the microbiota-gut-brain axis at all stages of life. Much recent work has implicated the gut microbiota in many conditions including autism, anxiety, obesity, schizophrenia, Parkinson's disease, and Alzheimer's disease. Animal models have been paramount in linking the regulation of fundamental neural processes, such as neurogenesis and myelination, to microbiome activation of microglia. Moreover, translational human studies are ongoing and will greatly enhance the field. Future studies will focus on understanding the mechanisms underlying the microbiota-gut-brain axis and attempt to elucidate microbial-based intervention and therapeutic strategies for neuropsychiatric disorders.
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http://dx.doi.org/10.1152/physrev.00018.2018DOI Listing
October 2019

Mid-life microbiota crises: middle age is associated with pervasive neuroimmune alterations that are reversed by targeting the gut microbiome.

Mol Psychiatry 2020 10 16;25(10):2567-2583. Epub 2019 May 16.

APC Microbiome Ireland, University College Cork, Cork, Ireland.

Male middle age is a transitional period where many physiological and psychological changes occur leading to cognitive and behavioural alterations, and a deterioration of brain function. However, the mechanisms underpinning such changes are unclear. The gut microbiome has been implicated as a key mediator in the communication between the gut and the brain, and in the regulation of brain homeostasis, including brain immune cell function. Thus, we tested whether targeting the gut microbiome by prebiotic supplementation may alter microglia activation and brain function in ageing. Male young adult (8 weeks) and middle-aged (10 months) C57BL/6 mice received diet enriched with a prebiotic (10% oligofructose-enriched inulin) or control chow for 14 weeks. Prebiotic supplementation differentially altered the gut microbiota profile in young and middle-aged mice with changes correlating with faecal metabolites. Functionally, this translated into a reversal of stress-induced immune priming in middle-aged mice. In addition, a reduction in ageing-induced infiltration of Ly-6C monocytes into the brain coupled with a reversal in ageing-related increases in a subset of activated microglia (Ly-6C) was observed. Taken together, these data highlight a potential pathway by which targeting the gut microbiome with prebiotics can modulate the peripheral immune response and alter neuroinflammation in middle age. Our data highlight a novel strategy for the amelioration of age-related neuroinflammatory pathologies and brain function.
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http://dx.doi.org/10.1038/s41380-019-0425-1DOI Listing
October 2020

Manipulation of gut microbiota blunts the ventilatory response to hypercapnia in adult rats.

EBioMedicine 2019 Jun 18;44:618-638. Epub 2019 Mar 18.

Department of Physiology, School of Medicine, College of Medicine & Health, University College Cork, Cork, Ireland; APC Microbiome Ireland, University College Cork, Cork, Ireland. Electronic address:

Background: It is increasingly evident that perturbations to the diversity and composition of the gut microbiota have significant consequences for the regulation of integrative physiological systems. There is growing interest in the potential contribution of microbiota-gut-brain signalling to cardiorespiratory control in health and disease.

Methods: In adult male rats, we sought to determine the cardiorespiratory effects of manipulation of the gut microbiota following a 4-week administration of a cocktail of antibiotics. We subsequently explored the effects of administration of faecal microbiota from pooled control (vehicle) rat faeces, given by gavage to vehicle- and antibiotic-treated rats.

Findings: Antibiotic intervention depressed the ventilatory response to hypercapnic stress in conscious animals, owing to a reduction in the respiratory frequency response to carbon dioxide. Baseline frequency, respiratory timing variability, and the expression of apnoeas and sighs were normal. Microbiota-depleted rats had decreased systolic blood pressure. Faecal microbiota transfer to vehicle- and antibiotic-treated animals also disrupted the gut microbiota composition, associated with depressed ventilatory responsiveness to hypercapnia. Chronic antibiotic intervention or faecal microbiota transfer both caused significant disruptions to brainstem monoamine neurochemistry, with increased homovanillic acid:dopamine ratio indicative of increased dopamine turnover, which correlated with the abundance of several bacteria of six different phyla.

Interpretation: Chronic antibiotic administration and faecal microbiota transfer disrupt gut microbiota, brainstem monoamine concentrations and the ventilatory response to hypercapnia. We suggest that aberrant microbiota-gut-brain axis signalling has a modulatory influence on respiratory behaviour during hypercapnic stress. FUND: Department of Physiology and APC Microbiome Ireland, University College Cork, Ireland.
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http://dx.doi.org/10.1016/j.ebiom.2019.03.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606895PMC
June 2019

Chronic intermittent hypoxia disrupts cardiorespiratory homeostasis and gut microbiota composition in adult male guinea-pigs.

EBioMedicine 2018 Dec 13;38:191-205. Epub 2018 Nov 13.

Department of Physiology, School of Medicine, College of Medicine & Health, University College Cork, Cork, Ireland; APC Microbiome Ireland, University College Cork, Cork, Ireland. Electronic address:

Background: Carotid body (peripheral oxygen sensor) sensitisation is pivotal in the development of chronic intermittent hypoxia (CIH)-induced hypertension. We sought to determine if exposure to CIH, modelling human sleep apnoea, adversely affects cardiorespiratory control in guinea-pigs, a species with hypoxia-insensitive carotid bodies. We reasoned that CIH-induced disruption of gut microbiota would evoke cardiorespiratory morbidity.

Methods: Adult male guinea-pigs were exposed to CIH (6.5% O at nadir, 6 cycles.hour) for 8 h.day for 12 consecutive days.

Findings: CIH-exposed animals established reduced faecal microbiota species richness, with increased relative abundance of Bacteroidetes and reduced relative abundance of Firmicutes bacteria. Urinary corticosterone and noradrenaline levels were unchanged in CIH-exposed animals, but brainstem noradrenaline concentrations were lower compared with sham. Baseline ventilation was equivalent in CIH-exposed and sham animals; however, respiratory timing variability, sigh frequency and ventilation during hypoxic breathing were all lower in CIH-exposed animals. Baseline arterial blood pressure was unaffected by exposure to CIH, but β-adrenoceptor-dependent tachycardia and blunted bradycardia during phenylephrine-induced pressor responses was evident compared with sham controls.

Interpretation: Increased carotid body chemo-afferent signalling appears obligatory for the development of CIH-induced hypertension and elevated chemoreflex control of breathing commonly reported in mammals, with hypoxia-sensitive carotid bodies. However, we reveal that exposure to modest CIH alters gut microbiota richness and composition, brainstem neurochemistry, and autonomic control of heart rate, independent of carotid body sensitisation, suggesting modulation of breathing and autonomic homeostasis via the microbiota-gut-brainstem axis. The findings have relevance to human sleep-disordered breathing.

Funding: The Department of Physiology, and APC Microbiome Ireland, UCC.
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http://dx.doi.org/10.1016/j.ebiom.2018.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306383PMC
December 2018

Differential functional selectivity and downstream signaling bias of ghrelin receptor antagonists and inverse agonists.

FASEB J 2019 01 18;33(1):518-531. Epub 2018 Jul 18.

Alimentary Pharmabiotic Centre (APC) Microbiome Ireland, University College Cork, Cork, Ireland.

The ghrelin receptor [growth hormone secretagogue receptor (GHSR)-1a] represents a promising pharmacologic target for the treatment of metabolic disorders, including obesity and cachexia, via central appetite modulation. The GHSR-1a has a complex pharmacology, highlighted by G-protein-dependent and -independent downstream signaling pathways and high basal constitutive activity. The functional selectivity and signaling bias of many GHSR-1a-specific ligands has not been fully characterized. In this study, we investigated the pharmacologic properties of ghrelin, MK-0677, L692,585, and [d-Lys3]-growth hormone-releasing peptide-6 (Dlys), JMV2959, and [d-Arg(1),d-Phe(5),d-Trp(7, 9),Leu(11)]-substance P (SP-analog). We investigated their effect on basal GHSR-1a constitutive signaling, ligand-directed downstream GHSR-1a signaling, functional selectivity, and signaling bias. Dlys behaved as a partial antagonist with a strong bias toward GHSR-1a-β-arrestin signaling, whereas JMV2959 acted as a full unbiased GHSR-1a antagonist. Moreover, the SP-analog behaved as an inverse agonist increasing G-protein-dependent signaling, but only at high concentrations, whereas, at low concentrations, the SP-analog attenuated β-arrestin-dependent signaling. Considering the limited success in the clinical development of GHSR-1a-targeted drugs so far, these findings provide a novel insight into the pharmacologic characteristics of GHSR-1a ligands and their signaling bias, which has important implications in the design of novel, more selective GHSR-1a ligands with predictable functional outcome and selectivity for preclinical and clinical drug development.-Ramirez, V. T., van Oeffelen, W. E. P. A., Torres-Fuentes, C., Chruścicka, B., Druelle, C., Golubeva, A. V., van de Wouw, M., Dinan, T. G., Cryan, J. F., Schellekens, H. Differential functional selectivity and downstream signaling bias of ghrelin receptor antagonists and inverse agonists.
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http://dx.doi.org/10.1096/fj.201800655RDOI Listing
January 2019

Strain differences in the susceptibility to the gut-brain axis and neurobehavioural alterations induced by maternal immune activation in mice.

Behav Pharmacol 2018 04;29(2 and 3-Spec Issue):181-198

APC Microbiome Institute.

There is a growing realization that the severity of the core symptoms of autism spectrum disorders and schizophrenia is associated with gastrointestinal dysfunction. Nonetheless, the mechanisms underlying such comorbidities remain unknown. Several genetic and environmental factors have been linked to a higher susceptibility to neurodevelopmental abnormalities. The maternal immune activation (MIA) rodent model is a valuable tool for elucidating the basis of this interaction. We induced MIA with polyinosinic-polycytidylic acid (poly I:C) at gestational day 12.5 and assessed behavioural, physiological and molecular aspects relevant to the gut-brain axis in the offspring of an outbred (NIH Swiss) and an inbred (C57BL6/J) mouse strain. Our results showed that the specific MIA protocol employed induces social deficits in both strains. However, alterations in anxiety and depression-like behaviours were more pronounced in NIH Swiss mice. These strain-specific behavioural effects in the NIH Swiss mice were associated with marked changes in important components of gut-brain axis communication: the endocrine response to stress and gut permeability. In addition, MIA-induced changes in vasopressin receptor 1a mRNA expression in the hypothalamus were observed in NIH Swiss mice only. Taken together, these data suggest that genetic background is a critical factor in susceptibility to the gut-brain axis effects induced by MIA.
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http://dx.doi.org/10.1097/FBP.0000000000000374DOI Listing
April 2018

Microbiota-related Changes in Bile Acid & Tryptophan Metabolism are Associated with Gastrointestinal Dysfunction in a Mouse Model of Autism.

EBioMedicine 2017 Oct 21;24:166-178. Epub 2017 Sep 21.

APC Microbiome Institute, University College Cork, Cork, Ireland; Department of Anatomy & Neuroscience, University College Cork, Cork, Ireland. Electronic address:

Autism spectrum disorder (ASD) is one of the most prevalent neurodevelopmental conditions worldwide. There is growing awareness that ASD is highly comorbid with gastrointestinal distress and altered intestinal microbiome, and that host-microbiome interactions may contribute to the disease symptoms. However, the paucity of knowledge on gut-brain axis signaling in autism constitutes an obstacle to the development of precision microbiota-based therapeutics in ASD. To this end, we explored the interactions between intestinal microbiota, gut physiology and social behavior in a BTBR TItpr3/J mouse model of ASD. Here we show that a reduction in the relative abundance of very particular bacterial taxa in the BTBR gut - namely, bile-metabolizing Bifidobacterium and Blautia species, - is associated with deficient bile acid and tryptophan metabolism in the intestine, marked gastrointestinal dysfunction, as well as impaired social interactions in BTBR mice. Together these data support the concept of targeted manipulation of the gut microbiota for reversing gastrointestinal and behavioral symptomatology in ASD, and offer specific plausible targets in this endeavor.
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http://dx.doi.org/10.1016/j.ebiom.2017.09.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652137PMC
October 2017

Molecular Bases of Brain Preconditioning.

Front Neurosci 2017 25;11:427. Epub 2017 Jul 25.

Department of Physiology and General Pathology, Medical Faculty, Lomonosov Moscow State UniversityMoscow, Russia.

Preconditioning of the brain induces tolerance to the damaging effects of ischemia and prevents cell death in ischemic penumbra. The development of this phenomenon is mediated by mitochondrial adenosine triphosphate-sensitive potassium ([Formula: see text]) channels and nitric oxide signaling (NO). The aim of this study was to investigate the dynamics of molecular changes in mitochondria after ischemic preconditioning (IP) and the effect of pharmacological preconditioning (PhP) with the [Formula: see text]-channels opener diazoxide on NO levels after ischemic stroke in rats. Immunofluorescence-histochemistry and laser-confocal microscopy were applied to evaluate the cortical expression of electron transport chain enzymes, mitochondrial [Formula: see text]-channels, neuronal and inducible NO-synthases, as well as the dynamics of nitrosylation and nitration of proteins in rats during the early and delayed phases of IP. NO cerebral content was studied with electron paramagnetic resonance (EPR) spectroscopy using spin trapping. We found that 24 h after IP in rats, there is a two-fold decrease in expression of mitochondrial [Formula: see text]-channels ( = 0.012) in nervous tissue, a comparable increase in expression of cytochrome c oxidase ( = 0.008), and a decrease in intensity of protein S-nitrosylation and nitration ( = 0.0004 and = 0.001, respectively). PhP led to a 56% reduction of free NO concentration 72 h after ischemic stroke simulation ( = 0.002). We attribute this result to the restructuring of tissue energy metabolism, namely the provision of increased catalytic sites to mitochondria and the increased elimination of NO, which prevents a decrease in cell sensitivity to oxygen during subsequent periods of severe ischemia.
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http://dx.doi.org/10.3389/fnins.2017.00427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524930PMC
July 2017

Quantitative analysis of mucosal oxygenation using ex vivo imaging of healthy and inflamed mammalian colon tissue.

Cell Mol Life Sci 2017 01 10;74(1):141-151. Epub 2016 Aug 10.

School of Biochemistry and Cell Biology, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland.

Colonic inflammation is associated with decreased tissue oxygenation, significantly affecting gut homeostasis. However, the crosstalk between O consumption and supply in the inflamed tissue are not fully understood. Using a murine model of colitis, we analysed O in freshly prepared samples of healthy and inflamed colon tissue. We developed protocols for efficient ex vivo staining of mouse distal colon mucosa with a cell-penetrating O sensitive probe Pt-Glc and high-resolution imaging of O concentration in live tissue by confocal phosphorescence lifetime-imaging microscopy (PLIM). Microscopy analysis revealed that Pt-Glc stained mostly the top 50-60 μm layer of the mucosa, with high phosphorescence intensity in epithelial cells. Measured O values in normal mouse tissue ranged between 5 and 35 μM (4-28 Torr), tending to decrease in the deeper tissue areas. Four-day treatment with dextran sulphate sodium (DSS) triggered colon inflammation, as evidenced by an increase in local IL6 and mKC mRNA levels, but did not affect the gross architecture of colonic epithelium. We further observed an increase in oxygenation, partial activation of hypoxia inducible factor (HIF) 1 signalling, and negative trends in pyruvate dehydrogenase activity and O consumption rate in the colitis mucosa, suggesting a decrease in mitochondrial respiration, which is known to be regulated via HIF-1 signalling and pyruvate oxidation rate. These results along with efficient staining with Pt-Glc of rat and human colonic mucosa reveal high potential of PLIM platform as a powerful tool for the high-resolution analysis of the intestinal tissue oxygenation in patients with inflammatory bowel disease and other pathologies, affecting tissue respiration.
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http://dx.doi.org/10.1007/s00018-016-2323-xDOI Listing
January 2017

Sex-dependent activity of the spinal excitatory amino acid transporter: Role of estrous cycle.

Neuroscience 2016 10 25;333:311-9. Epub 2016 Jul 25.

Department of Anatomy and Neuroscience, University College Cork, Ireland; APC Microbiome Institute, University College Cork, Ireland. Electronic address:

Females are more likely to experience visceral pain than males, yet mechanisms underlying this sex bias are not fully elucidated. Moreover, pain sensitivity can change throughout the menstrual cycle. Alterations in the glutamatergic system have been implicated in several pain-disorders; however, whether these are sex-dependent is unclear. Thus, we aimed to investigate sex differences in the spinal cord glutamate uptake and how it varies across the estrous cycle. The activity of the glutamate transporters, excitatory amino acid transporters (EAATs) was assessed using an ex vivo aspartate radioactive uptake assay in the lumbosacral spinal cord in Sprague-Dawley male and female rats. The gene expression of EAATs, glutamate receptor subunits NR1 and NR2B and the estrogen receptors ERα & ERβ in the spinal cord were also analyzed. EAAT activity was lower in females, particularly during the estrus phase, and this was the only cycle stage that was responsive to the pharmacological effects of the EAATs activator riluzole. Interestingly, EAAT1 mRNA expression was lower in high-estrogen and high-ERα states compared to diestrus in females. We conclude that the Spinal EAAT activity in females is different to that in males, and varies across the estrous cycle. Furthermore, the expression levels of estrogen receptors also showed a cycle-dependent pattern that may affect EAATs function and expression.
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http://dx.doi.org/10.1016/j.neuroscience.2016.07.036DOI Listing
October 2016

Negative allosteric modulation of the mGlu7 receptor reduces visceral hypersensitivity in a stress-sensitive rat strain.

Neurobiol Stress 2015 4;2:28-33. Epub 2015 Apr 4.

Laboratory of Neurogastroenterology, Alimentary Pharmabiotic Centre, Biosciences Institute, University College Cork, Ireland; Department of Anatomy and Neuroscience, University College Cork, Ireland.

Glutamate, the main excitatory neurotransmitter in the central nervous system, exerts its effect through ionotropic and metabotropic receptors. Of these, group III mGlu receptors (mGlu 4, 6, 7, 8) are among the least studied due to a lack of pharmacological tools. mGlu7 receptors, the most highly conserved isoform, are abundantly distributed in the brain, especially in regions, such as the amygdala, known to be crucial for the emotional processing of painful stimuli. Visceral hypersensitivity is a poorly understood phenomenon manifesting as an increased sensitivity to visceral stimuli. Glutamate has long been associated with somatic pain processing leading us to postulate that crossover may exist between these two modalities. Moreover, stress has been shown to exacerbate visceral pain. ADX71743 is a novel, centrally penetrant, negative allosteric modulator of mGlu7 receptors. Thus, we used this tool to explore the possible involvement of this receptor in the mediation of visceral pain in a stress-sensitive model of visceral hypersensitivity, namely the Wistar Kyoto (WKY) rat. ADX71743 reduced visceral hypersensitivity in the WKY rat as exhibited by increased visceral sensitivity threshold with concomitant reductions in total number of pain behaviours. Moreover, AD71743 increased total distance and distance travelled in the inner zone of the open field. These findings show, for what is to our knowledge, the first time, that mGlu7 receptor signalling plays a role in visceral pain processing. Thus, negative modulation of the mGlu7 receptor may be a plausible target for the amelioration of stress-induced visceral pain where there is a large unmet medical need.
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http://dx.doi.org/10.1016/j.ynstr.2015.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4721404PMC
February 2016

Prenatal stress-induced alterations in major physiological systems correlate with gut microbiota composition in adulthood.

Psychoneuroendocrinology 2015 Oct 17;60:58-74. Epub 2015 Jun 17.

Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland; Department of Anatomy & Neuroscience, University College Cork, Cork, Ireland. Electronic address:

Early-life adverse experiences, including prenatal stress (PNS), are associated with a higher prevalence of neurodevelopmental, cardiovascular and metabolic disorders in affected offspring. Here, in a rat model of chronic PNS, we investigate the impact of late gestational stress on physiological outcomes in adulthood. Sprague-Dawley pregnant dams were subjected to repeated restraint stress from embryonic day 14 to day 20, and their male offspring were assessed at 4 months of age. PNS induced an exaggeration of the hypothalamic-pituitary-adrenal (HPA) axis response to stress, as well as an elevation of blood pressure and impairment of cognitive function. Altered respiratory control was also observed, as demonstrated by increased variability in basal respiratory frequency and abnormal frequency responses to both hypoxic and hypercapnic challenges. PNS also affected gastrointestinal neurodevelopment and function, as measured by a decrease in the innervation density of distal colon and an increase in the colonic secretory response to catecholaminergic stimulation. Finally, PNS induced long lasting alterations in the intestinal microbiota composition. 16S rRNA gene 454 pyrosequencing revealed a strong trend towards decreased numbers of bacteria in the Lactobacillus genus, accompanied by elevated abundance of the Oscillibacter, Anaerotruncus and Peptococcus genera in PNS animals. Strikingly, relative abundance of distinct bacteria genera significantly correlated with certain respiratory parameters and the responsiveness of the HPA axis to stress. Together, these findings provide novel evidence that PNS induces long-term maladaptive alterations in the gastrointestinal and respiratory systems, accompanied by hyper-responsiveness to stress and alterations in the gut microbiota.
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http://dx.doi.org/10.1016/j.psyneuen.2015.06.002DOI Listing
October 2015

Metabotropic Glutamate Receptors in Central Nervous System Diseases.

Curr Drug Targets 2016 ;17(5):538-616

Department of Anatomy & Neuroscience, University College Cork, Cork, Ireland.

Extensive research over the past thirty years has demonstrated a vital role for metabotropic glutamate (mGlu) receptors in the major functions of the central nervous system (CNS). A wealth of preclinical studies provide evidence that pharmacological targeting of mGlu receptors can effectively attenuate the development of symptoms and progression of many CNS disorders in animal models. In this review we summarize the current knowledge on the involvement of mGlu receptors in the pathophysiology of neuropsychiatric disorders (schizophrenia, depression, anxiety and cognitive disorders, pain perception and addiction), as well as neurodegenerative (Alzheimer's, Huntington's and Parkinson's diseases) and neurodevelopmental (fragile X syndrome and autism spectrum disorders) diseases. We further emphasize the therapeutic potential of mGlu receptors' pharmacological modulators in these diseases, describe the results of clinical trials with these compounds and discuss the potential sources of translational difficulties.
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http://dx.doi.org/10.2174/1389450116666150316224011DOI Listing
December 2016

Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling.

Exp Cell Res 2015 Jan 15;330(1):13-28. Epub 2014 Oct 15.

School of Biochemistry and Cell Biology, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland.

Changes in availability and utilisation of O2 and metabolic substrates are common in ischemia and cancer. We examined effects of substrate deprivation on HIF signalling in PC12 cells exposed to different atmospheric O2. Upon 2-4h moderate hypoxia, HIF-α protein levels were dictated by the availability of glutamine and glucose, essential for deep cell deoxygenation and glycolytic ATP flux. Nuclear accumulation of HIF-1α dramatically decreased upon inhibition of glutaminolysis or glutamine deprivation. Elevation of HIF-2α levels was transcription-independent and associated with the activation of Akt and Erk1/2. Upon 2h anoxia, HIF-2α levels strongly correlated with cellular ATP, produced exclusively via glycolysis. Without glucose, HIF signalling was suppressed, giving way to other regulators of cell adaptation to energy crisis, e.g. AMPK. Consequently, viability of cells deprived of O2 and glucose decreased upon inhibition of AMPK with dorsomorphin. The capacity of cells to accumulate HIF-2α decreased after 24h glucose deprivation. This effect, associated with increased AMPKα phosphorylation, was sensitive to dorsomorphin. In chronically hypoxic cells, glutamine played no major role in HIF-2α accumulation, which became mainly glucose-dependent. Overall, the availability of O2 and metabolic substrates intricately regulates HIF signalling by affecting cell oxygenation, ATP levels and pathways involved in production of HIF-α.
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http://dx.doi.org/10.1016/j.yexcr.2014.10.005DOI Listing
January 2015

The mouse cyclophosphamide model of bladder pain syndrome: tissue characterization, immune profiling, and relationship to metabotropic glutamate receptors.

Physiol Rep 2014 27;2(3):e00260. Epub 2014 Mar 27.

Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland.

Abstract Painful bladder syndrome/Interstitial cystitis (PBS/IC) is a chronic disorder characterized clinically by recurring episodes of pelvic pain and increased urination frequency, significantly impairing patients' quality of life. Despite this, there is an unmet medical need in terms of effective diagnostics and treatment. Animal models are crucial in this endeavor. Systemic chronic administration of cyclophosphamide (CYP) in mice has been proposed as a relevant preclinical model of chronic bladder pain. However, molecular mechanisms underlying the pathogenesis of this model are lacking. Here, we show that mice, subjected to repetitive systemic injections of CYP, developed mild inflammatory response in bladder tissue characterized by submucosal edema, moderate increase in proinflammatory cytokine gene expression, and mastocytosis. No signs of massive inflammatory infiltrate, tissue hemorrhages, mucosal ulcerations and urothelium loss were observed. Instead, CYP treatment induced urothelium hyperplasia, accompanied by activation of proliferative signaling cascades, and a decrease in the expression of urothelium-specific markers. Metabotropic glutamate (mGlu) receptors have been implicated in chronic pain disorders. CYP administration induced differential changes in mGlu receptors mRNA levels in bladder tissue, without affecting gene expression at spinal cord level, pointing to the potential link between peripheral mGlu receptors and inflammation-induced bladder malfunction and hyperalgesia. Taken together, these data indicate that chronic CYP treatment in mice is a model of PBS mostly relevant to the major, nonulcerative subtype of the syndrome, characterized by a relatively unaltered mucosa and a sparse inflammatory response. This model can help to elucidate the pathogenetic mechanisms of the disease.
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http://dx.doi.org/10.1002/phy2.260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4002240PMC
April 2014

GABA receptors in the bladder and bowel: therapeutic potential for positive allosteric modulators?: Commentary on Kalinichev et al., Br J Pharmacol 171: 995-1006.

Br J Pharmacol 2015 Sep;172(18):4588-4590

Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland.

Linked Article: This article is a Commentary on Kalinichev M, Palea S, Haddouk H, Royer-Urios I, Guilloteau V, Lluel P, Schneider M, Saporito M and Poli S (2014). ADX71441, a novel, potent and selective positive allosteric modulator of the GABAB receptor, shows efficacy in rodent models of overactive bladder. Br J Pharmacol 171: 995-1006. doi: 10.1111/bph.12517.
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http://dx.doi.org/10.1111/bph.12617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4562517PMC
September 2015

Toll-like receptor 4 regulates chronic stress-induced visceral pain in mice.

Biol Psychiatry 2014 Aug 11;76(4):340-8. Epub 2013 Nov 11.

Laboratory of NeuroGastroenterology, Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland; Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland. Electronic address:

Background: Functional gastrointestinal disorders, which have visceral hypersensitivity as a core symptom, are frequently comorbid with stress-related psychiatric disorders. Increasing evidence points to a key role for toll-like receptor 4 (TLR4) in chronic pain states of somatic origin. However, the central contribution of TLR4 in visceral pain sensation remains elusive.

Methods: With pharmacological and genetic approaches, we investigated the involvement of TLR4 in the modulation of visceral pain. The TLR4-deficient and wild-type mice were exposed to chronic stress. Visceral pain was evaluated with colorectal distension. Protein expression levels for TLR4, Cd11b, and glial fibrillary acidic protein (glial cells markers) were quantified in the lumbar region of the spinal cord, prefrontal cortex (PFC), and hippocampus. To evaluate the effect of blocking TLR4 on visceral nociception, TAK-242, a selective TLR4 antagonist, was administered peripherally (intravenous) and centrally (intracerebroventricular and intra-PFC) (n = 10-12/experimental group).

Results: The TLR4 deficiency reduced visceral pain and prevented the development of chronic psychosocial stress-induced visceral hypersensitivity. Increased expression of TLR4 coupled with enhanced glia activation in the PFC and increased levels of proinflammatory cytokines were observed after chronic stress in wild-type mice. Administration of a TLR4 specific antagonist, TAK-242, attenuated visceral pain sensation in animals with functional TLR4 when administrated centrally and peripherally. Moreover, intra-PFC TAK-242 administration also counteracted chronic stress-induced visceral hypersensitivity.

Conclusions: Our results reveal a novel role for TLR4 within the PFC in the modulation of visceral nociception and point to TLR4 as a potential therapeutic target for the development of drugs to treat visceral hypersensitivity.
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http://dx.doi.org/10.1016/j.biopsych.2013.11.004DOI Listing
August 2014

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling.

Biochim Biophys Acta 2014 Jan 23;1837(1):51-62. Epub 2013 Jul 23.

Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland. Electronic address:

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O2 consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca(2+) or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.
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http://dx.doi.org/10.1016/j.bbabio.2013.07.008DOI Listing
January 2014

Chronic hypoxia leads to a glycolytic phenotype and suppressed HIF-2 signaling in PC12 cells.

Biochim Biophys Acta 2013 Jun 24;1830(6):3553-69. Epub 2013 Feb 24.

Biochemistry Department, University College Cork, Cork, Ireland.

Background: Along with other regulators of cell metabolism, hypoxia-inducible factors HIF-1 and HIF-2 differentially regulate cell adaptation to hypoxia. Switches in HIF-1/HIF-2 signaling in chronic hypoxia have not been fully investigated.

Methods: Proliferation, viability, apoptosis, neuronal and bioenergetic markers, mitochondrial function, respiration, glycolysis, HIF signalling, responses to O2 and glucose deprivation (OGD) were examined using tumor PC12 and SH-SY5Y cells continuously grown at 3% O2.

Results: Hypoxic PC12 cells (H-cells) exhibit reduced proliferation and histone H4 acetylation, NGF-independent differentiation, activation of AMPK, inhibition of Akt, altered mitochondria and response to NGF. Cellular cytochrome c is increased with no effect on apoptosis. Reduction in respiration has minor effect on cellular ATP which is maintained through activated uptake (GLUT1) and utilization (HK2, PFK2) of glucose. H-cells exhibit resistance to OGD linked to increased glycogen stores. HIF-2alpha protein is decreased without changes in mRNA. Unlike HIF-1alpha, HIF-2alpha is not stabilized pharmacologically or by O2 deprivation. Capacity for HIF-2alpha stabilization is partly restored when H-cells are cultured at normoxia. In low-respiring SH-SY5Y cells cultured under the same conditions HIF-2alpha stabilization and energy budget are not affected.

Conclusions: In chronically hypoxic PC12 cells glycolytic energy budget, increased energy preservation and low susceptibility to OGD are observed. HIF-2alpha no longer orchestrates adaptive responses to anoxia.

General Significance: Demonstrated switch in HIF-1/HIF-2 signaling upon chronic hypoxia can facilitate cell survival in energy crisis, by regulating balance between energy saving and decrease in proliferation, on one hand and active cell growth and tumor expansion, on the other.
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http://dx.doi.org/10.1016/j.bbagen.2013.02.016DOI Listing
June 2013

Long-term normalization of blood pressure in SHR and 1-kidney 1-clip rats by synthetic precursor of stable PAF analogue without systemic effects in normotensive rats.

Pathophysiology 2011 Apr;18(2):151-7

Institute of Organic Chemistry and Technology, Moscow, Russia.

This study characterized the actions of the newly synthesized PAF precursor 1-hexadecyl-2-alkylcarbamoyl-glycerol (HAG) on blood pressure (BP) in male spontaneously hypertensive rats (SHR), SHR-stroke prone (SHRSP) and Wistar rats with 1-kidney 1-clip (1K1C) renovascular hypertension used as experimental models of human primary and secondary hypertension. Systolic blood pressure (SBP) in the tail artery and mean arterial pressure (MAP) in the abdominal aorta were measured by tail plethysmography and invasive pressure transducer, respectively. Intravenous treatment with 1mg/kg HAG in SHR resulted in a rapid decline of MAP from 151±4 to 127±4mmHg in 50min (p<0.001) that was maintained for 24h after injection (128±5mmHg, p<0.01). We also observed a profound hypotensive effect of HAG in SHRSP but not in normotensive Wistar rats. In 1K1C rats, the magnitude of the BP decline evoked by HAG was correlated with MAP measured before drug administration (R=0.74, p<0.005). In 1K1C rats with SBP>140mmHg, 5mg/kg/48h HAG, given orally for 14 days, decreased SBP by 20-30mmHg without an increase in the death rate and other adverse effects. Thus, our results show that intravenous and oral administration of HAG led to a long-lasting reduction of BP in experimental models of primary and secondary hypertension. In contrast to PAF and its derivatives, the hypotensive action of HAG was preserved for 24h after a single administration, was absent in normotensive animals, and was not accompanied by visible side-effects, at least during 2 weeks of treatment.
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http://dx.doi.org/10.1016/j.pathophys.2010.08.001DOI Listing
April 2011