Psychiatry - Doctoral Theses

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The role of the gut microbiota in the function and integrity of brain barriers
(University College Cork, 2023) Knox, Emily G.; Cryan, John; O'Driscoll, Caitriona M.; Rodriguez Aburto, Maria; Clarke, Gerard
The barriers of the brain help maintain an optimal environment for central nervous system (CNS) function and homeostasis. There are many instances such as during age related disorders and neuropsychiatric disorders in which these barriers are under threat. Importantly, the gut-microbiota can interact with the brain barriers, thereby altering barrier physiology and communication with the brain. This bidirectional communication is known as the microbiota-gut-brain axis. Modulation of the gut microbiota can thereby alter functions in the brain, potentially through influence of the brain barriers. In this thesis we focus on the interface of both brain barriers; the blood-cerebrospinal fluid barrier (CSF) and blood-brain barrier (BBB) in microbiota-gut-brain axis communication. In particular, using germ-free mice which lack a gut microbiota, we investigated the contributing role of the gut microbiota in regulation of the blood-CSF barrier. Following confocal microscopy for visualization of the tight junction protein, ZO-1, germ-free mice were found to have a disruption of the tight junction protein networks in isolated choroid plexus tissue. Similar assessment of the choroid plexus capillaries revealed no difference between germ-free and conventional mice. Since microbial metabolites are some of the known contributing factors of the gut microbiota’s manipulation of the barriers, we next investigated the effects of physiological concentrations of short chain fatty acids (SCFAs), butyrate and propionate, with and without induced barrier dysfunction (LPS) on the actin cytoskeleton and tight junction protein dynamics using an in vitro BBB model. We found that the butyrate and propionate altered filamentous actin directionality, increased tight junction protein spikes, and protected from LPS induced decrease in mitochondrial footprint and barrier integrity measured by trans-endothelial electrical resistance (TEER). While we do not yet understand the dynamics of the BBB following acute stress, we know that microbial metabolites can interact with BBB function, so we next assessed the potential for acute-stress induced metabolites to regulate BBB integrity using the same in vitro model. Following TEER assessment of a broad range of concentrations, we found that microbial metabolites from different classes; SCFAs, bile acids, and tryptophan metabolites impact BBB integrity. Specifically, butyrate, propionate, acetate, 3β-hydroxy-5-cholenic acid, and tryptamine exerted protective effects while acetate and cholic acid exerted detrimental effects at various concentrations. Lastly, since diet is a major modulator of the gut microbiota and especially in infancy, we assessed the impact of infant formulations fermented with probiotic strains of Bifidobacterium uniquely isolated from infant stool samples for potential effects on BBB integrity. Cell-free supernatants from the fermentation process which would contain a pool of microbial metabolites were then collected and exposed to the same in vitro model for assessment of barrier integrity with and without LPS disruption. We found that in each of the four infant formulations, there was protection from LPS disruption following fermentation with some of the Bifidobacterium strains. Overall, these results provide valuable insight into the interphase of brain barriers in microbiota-gut-brain axis communication. We highlight the role of another brain barrier in this interface, the blood-CSF barrier and further elucidate the microbial metabolite interaction in BBB physiology. Further, using two of the major modulators of the gut microbiota, stress and diet, we reveal that there is scope for the impacted metabolites or metabolic pools to influence brain barrier interactions.
Unravelling the role of tryptophan metabolism in the microbiota-gut-brain axis: focus on the effects of stress and exercise
(University College Cork, 2023) Gheorghe, Cassandra Elise; Clarke, Gerard; Cryan, John; Dinan, Timothy G.; FEANS; Léandre Pourcelot
The involvement of the microbiota-gut-brain axis communication in brain function and behaviour has enabled a paradigm shift in neuroscience, neurogastroenterology and psychiatric research. Two key routes of this bidirectional communication are impaired in stress-related disorders: the hypothalamic-pituitary-adrenal axis and tryptophan metabolism. This crosstalk has been widely characterised following chronic stress, in stress-related disorders and gastrointestinal disorders comorbid with psychological distress. The acute transitory reaction to an acute stressor and the associated adaptation over time, however, is not well understood. In this work, we investigated this neglected topic by evaluating the response to a psychological and a physical s stress exposure in terms host and microbial tryptophan metabolism and other pillars of the microbiota-gut-brain axis. For this purpose, we used well-recognised pre-clinical models (germ-free and antibiotic-treated mice) to decipher the role of gut microbes in the regulation of tryptophan metabolism and availability at baseline and following hypothalamic-pituitary-adrenal axis activation. We found that a 15-min acute stressor was sufficient to upregulate colonic 5-HT concentrations in conventional and re-colonised male mice while serotonin level was unaltered in GF male mice. We have identified novel region- and sex- dependent effects of the microbiota in modulating the expression of host rate-limiting enzyme of tryptophan metabolism following acute stress at both terminals of the gut-brain axis communication network (Chapter 2). We found that acute stress induced functional alterations visible as increased intestinal permeability in the ileum (ex vivo and in vitro) depending on time of day. In the colon, alterations in the rate-limiting enzyme of tryptophan breakdown towards serotonin synthesis, TPH-1, was reduced by stress in conventional mice but increased in antibiotic treated mice following stress (Chapter 3). Furthermore, both microbial and host tryptophan metabolism were altered in the caecum of conventional mice following an acute stressor, which was disturbed in germ-free mice and partially restored following colonisation of germ-free animals (Chapter 3). Then, we assessed in sedentary humans transient and adaptive changes to exercise along the microbiota-gut-brain axis at different exercise intensities. We discovered intensity-dependent effects on various pillars of the microbiota-gut-brain axis. While high-intensity training increased the cortisol awakening response chronically, it also led to an increase in peripheral serotonin level directly following a maximal exhaustion test. Light-intensity exercise lead to marked compositional alterations at the species level. Lastly, our work provides the first piece of evidence that the relative abundance of exercise-responsive bacteria with saccharolytic potential decreases with exercise intensity. Together, these results contribute to a deeper comprehension of the host-microbe dialogue driving the spatial and temporal dynamics of the physiological response and adaptation to various types of stress exposure. These observations open future research avenues and encourages a move towards the integrated physiological perspectives essential for the development of precision treatment options in stress-related disorders.
From youth to old age: therapeutic potential of microbiota interventions in the gut-brain-axis
(University College Cork, 2022-08-17) Minuto, Chiara; Dinan, Timothy G.; Cryan, John; Clarke, Gerard
There is a growing recognition of the involvement of the gut microbiota in the regulation of certain physiological and metabolic parameters across the life cycle of animals. A greater understanding of the microbiota changes throughout the life cycle may provide a novel therapeutic target for treating various age-related disorders from autism in childhood to dementia in old age. The increase in the elderly population with a rapid rise in dementia, cardiovascular and metabolic disease, leads to the growing demand for new interventions to decelerate the senescent decline. However, if the microbiota is to become a therapeutic target a far greater understanding of the structural changes which take place throughout the lifespan is required. In this thesis, we focus attention on microbiota-targeted interventions in young, middle age and old animals. The studies examine behaviour, physiology, and metabolism, exploring effects both in the periphery and in the brain, across a range of treatments from prebiotics to polyphenols. We highlight the physiological differences between the various age groups. Intestinal permeability and neuronal plasticity were the only modifiable parameters in the elderly. In younger animal prebiotics impact metabolism and inflammation. Polyphenols rich diets (grape pomace and rafuma) significantly alter intestinal permeability reversing the damage to the intestinal barrier due to ageing. The effect was not directly linked to changes in microbial diversity of the gut and did not significantly ameliorate the impaired immune system of the aged animals’ intestine or hippocampus. Only a significant increase in the number of dendritic cells emerged in conjunction with a reduced level of TNF-α in the blood. Neither did the polyphenols reverse some of the behavioural deficits associated with ageing. Cumulatively, these data show the specific effect of polyphenols in the elderly. We next investigated whether inulin, the most widely studied prebiotics, could improve host metabolism in middle-aged mice. We tested 52 metabolic markers involved in hypothalamic-pituitary-adrenal (HPA) axis regulation, cholesterol and glucose homeostasis and immune regulation. Interestingly, the liver, the brain and the mesenteric fat did not show any modification in middle-age animals. Inulin had a greater impact in young animals and stimulated the lipid and glucose metabolism, increasing the level of Sirtuin1, a fundamental metabolic sensor, without impacting transcriptional factor changes in the liver. The study also uncovered a range of changes in the HPA. In fact, inulin decreased the level of the glucocorticoids receptors (Nr3c1) and corticotropin-releasing hormone (Crhr1) receptors in the amygdala of young mice. In an attempt to understand the mechanisms through which microbiota-targeted interventions exert the anti-aging effects, we used a model of premature ageing (ApoE deficient mice). The effect of two different strains of lactobacillus (L.reuteri and L.mucosae) and two prebiotics derived from plants (plant sterol esters and oat b- glucan) were examined. Neural plasticity was restored in the Prefrontal cortex after 24 weeks of prebiotic administration and the level of claudin 5 increased in the hippocampus following consumption of L. mucosae. Overall, these results provide novel insights regarding the impact of a portfolio of microbiota-targeted interventions on age-related alterations in physiology and brain function and suggest the importance of using target-specific therapeutic options during particular time windows for maximum gain.
The microbiota-gut-brain axis in social anxiety disorder
(University College Cork, 2021-07) Butler, Mary I.; Dinan, Timothy G.; Clarke, Gerard; O'Mahony, Siobhain M.
The past decade has seen huge interest in the role of microbiota-gut-brain (MGB) axis in psychiatric disorders. Significant preclinical efforts have been made to elucidate the role of the gut microbiome in the stress response, and there is an ever-growing body of evidence demonstrating the effect of gut microbiome modulation on behaviour in various animal models of anxiety and depression. Additionally, studies in healthy human volunteers have generated hope that microbiome-based interventions may improve mood and anxiety symptoms. Despite this, the MGB axis remains largely unexplored in patients with clinical anxiety disorders, such as social anxiety disorder (SAD). Indeed, investigation of the neurobiological basis of this and other clinical anxiety conditions is limited, and these disorders remain poorly understood. To this end, we hypothesized that the gut microbiota would be altered in those with SAD, and that gut barrier dysfunction would be evident. We proposed that physiological systems of relevance in MGB communication, including neuroendocrine, immune, and tryptophan-kynurenine pathways would show differences compared to controls. In this thesis, we demonstrate that the gut microbiome is compositionally and functionally altered in patients with SAD, and that this patient group have compromised intestinal permeability. We demonstrate that SAD is associated with differences in various systems involved in MGB communication. We report elevated kynurenic acid (KYNA) levels and an increased KYNA/Kynurenine ratio in our patient group. Additionally, SAD patients show lower levels of the anti-inflammatory cytokine, interleukin-10, along with various neuroendocrine alterations including lower oxytocin levels and differences in the cortisol awakening response, chronic cortisol concentrations and morning salivary alpha amylase levels. Taken together, our results raise the possibility that the MGB axis may represent an important aetiological node and potential therapeutic target for this early-onset, chronic disorder. Our work supports the need for larger, longitudinal studies to further explore the role of the MGB axis in clinical anxiety disorders. In addition, we report on a longitudinal study assessing the impact of a dietary change involving the consumption of unpasteurised dairy, on the gut microbiome of healthy volunteers. We demonstrate that intake of unpasteurised dairy is associated with significant increases in Lactobacillus, a psychobiotic bacterial genus which is recognised as having anxiolytic and antidepressant effects. This work supports the possibility that dietary change may have therapeutic potential in psychiatric conditions.
Gut microbes and brain function
(University College Cork, 2021-04-30) Wiley, Niamh; Stanton, Catherine; Ross, R. Paul; Cryan, John; Dinan, Timothy G.; Science Foundation Ireland; Seventh Framework Programme
The gut-brain-microbiota axis comprises an extensive communication network between the brain, the gut, and the microbiota residing there. If the composition or the diversity of the gut microbiota is impaired, this can have negative consequences for host health and has been associated with disorders such as obesity, diabetes, inflammatory diseases, and neuropsychiatric illnesses including anxiety and depression. Therefore, much research effort in recent years has focused on understanding the potential of targeting the intestinal microbiota to prevent and treat such disorders. Preclinical data strongly support the view that microbe manipulation using probiotics and prebiotics to enhance the host-microbe symbiotic relationship has great potential in the future prevention and treatment of such disorders. Therefore, this study aimed to identify novel probiotics by screening gut-derived lactobacilli for bioactive metabolite production, namely GABA and serotonin, identifying one Lactobacillus plantarum GABA producer and two Lactobacillus paracasei serotonin producers which were subsequently characterised and assessed for probiotic characteristics. Enhanced whole genome sequencing identified the trp operon involved in tryptophan and serotonin metabolic pathways, antimicrobial activity including Enterolysin A and Carnocin CP52, as well as genes promoting acid tolerance and adhesion capability such as dlt and fbp. The work described here also explores the influence that environmental factors and diet have on the microbiome and the subsequent effect on anxiety and stress-related behaviour in mouse models and an ecologically relevant model, namely wild caught great tit birds. Chapter 4 explores the effect of environment on the gut microbiota by exposing high anxiety and normal anxiety phenotype mice to an enriched versus standard environment, revealing improved anxiety-like behaviour following environmental enrichment. However, no effect was observed on alpha diversity while differences in beta diversity were based on mouse phenotype and not environment. Chapter 5 investigated the impact of diet on the gut microbiome in wild caught great tit birds, and demonstrates that a diet low in fat, protein and fibre results in lower alpha diversity compared to a high fat, protein and fibre content diet, while several compositional differences were also observed, including a higher abundance of Bacteroidetes and Proteobacteria following administration of an insect diet. Furthermore, the data presented herein outline the potential of harnessing the gut microbiota to improve metabolic and neuropsychological health through probiotic and prebiotic intervention in preclinical and clinical trials, respectively. The data presented in Chapter 6 outline a potential role of metabolic- and neuroactive-microbial metabolite production in the modulation of diet-induced metabolic dysfunction, including abnormal behaviour. Intervention with GABA-producing Lactobacillus brevis strains attenuated several abnormalities associated with metabolic dysfunction, causing a reduction in the accumulation of mesenteric adipose tissue, increased insulin secretion following glucose challenge, improved plasma cholesterol clearance, and reduced despair-like behaviour and basal corticosterone production during the forced swim test. Chapter 7 investigated an alternative method of gut microbiota manipulation, namely prebiotic administration with polydextrose in adults for four weeks which resulted in a modest improvement in cognitive flexibility and an increased ability for sustained attention in this double-blind, randomised, placebo-controlled clinical trial. Although there was no change in microbial diversity, abundance of Ruminiclostridium 5 significantly increased after polydextrose supplementation compared to placebo. Therefore, the results presented herein indicate that probiotic and prebiotic intervention could modulate gut-to-brain communication and benefit metabolic and neuropsychological health.

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