Psychiatry - Doctoral Theses

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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.
The neurobiological effects of naturally-derived polyphenols and phospholipids in cellular & animal models of stress
(University College Cork, 2019-12-20) Donoso, Francisco; Cryan, John; Dinan, Timothy G.; Stanton, Catherine; Science Foundation Ireland; Department of Agriculture, Food and the Marine, Ireland
The molecular and cellular basis of stress neurobiology remain an important research question in clinical science. Indeed, stress-related mental disorders, including depression and anxiety, are currently a major public health concern. Thus, improving our knowledge about the pathophysiology of these neuropsychiatric disorders may enable the development of novel strategies for their treatment and prevention. On the other hand, the inefficacy of currently available therapies for various stress-related disorders, and the numerous side effects that accompany these treatments, have strengthened the search for less invasive strategies with fewer negative side effects. In this regard, the emerging and compelling evidence for nutrition as a potential therapeutic avenue for the treatment of mental disorders suggests that changes in diet are a viable strategy for improving mental health and treating stress-related psychiatric disorders. Moreover, there is considerable evidence suggesting that certain natural compounds available in diet have a therapeutic potential to improve mental health and disease. For instance, naturally occurring phytochemicals, namely polyphenols, are molecular compounds found in different plant sources, such as vegetables and fruits. Also, phospholipids are a class of lipid that comprise a major component of all cell membranes, specially concentrated in lean meat and dairy products. Both polyphenols and phospholipids have demonstrated interesting beneficial effects for human health. However, their therapeutic potential to act prophylactically against the detrimental effects of neuropsychiatric disorders have just begun to be taken seriously. Therefore, in this thesis we have tested the hypothesis that polyphenols and/or phospholipids could improve behavioural and neurobiological outcomes in cellular and animal models of stress. Further, we provide evidence that polyphenols and phospholipids exert neuroprotective effects against the cytotoxicity produced by corticosterone, the main rodent stress hormone, in cortical neurons. Specifically, we have elucidated the potential mechanisms underlying polyphenol-mediated neuroprotection in vitro, and demonstrated that phospholipid exposure positively impacts on neurodevelopmental processes, such as proliferation and differentiation of cultured neural progenitor cells. In addition, we confirmed the therapeutic potential of a dietary intervention with polyphenols by detecting its capacity to reverse depressive- and anxiety-like behaviours induced in a rat model of early-life stress. Moreover, we demonstrated potential implications to modulate BDNF-dependent recovery, regulation of the HPA axis and the microbiota-gut-brain axis in polyphenol-mediated behavioural improvement. Taken together, our findings support the therapeutic potential of polyphenols for stress-related mental disorders, and we further provide evidence for the possible mechanisms by which they may exert these effects. On the other hand, our data reveal that the novel neuromodulatory potential of phospholipids in vitro does not correlate with their inefficacy in attenuating chronic stress-induced behavioural impairment in mice. Nevertheless, these findings contribute to an exciting and growing body of research suggesting that nutritional interventions may have an important role to play in the treatment of stress-related psychiatric conditions.
Dairy phospholipids: analysis, enrichment and biological application
(University College Cork, 2017) Barry, Kate M.; Dinan, Timothy G.; Kelly, Philip; Stanton, Catherine; Department of Agriculture, Food and the Marine
Dairy phospholipids (PLs) are complex polar lipids with a unique amphiphilic nature underpinning their structural integrity as a membrane surrounding milk fat globules- the milk fat globule membrane (MFGM). The techno-functional capabilities, nutritional benefits and putative health associations of dairy PLs have garnered considerable interest over the last number of decades and thus significant research has focused on the analysis, isolation and application of these bioactive and functional compounds. Development of a robust and reliable analytical method for efficient recovery and accurate quantitative determination of dairy PLs was achieved in the course of comparing different lipid extraction protocols and optimisation of high performance liquid chromatography (HPLC) procedures. Reappraisal of current lipid extraction procedures based on PL recovery determined Folch as the most effective method for PL analysis with a 1.9-fold and 2.5-fold increase in PL recovery compared with Röse Gottlieb and a modified Folch, respectively. Concomitantly, optimisation of a HPLC method coupled to a charged aerosol detection system (CAD) yielded a two-fold increase in PL recovery compared to the original method. Alteration of the HPLC elution program maximised PL peak separation, and pH adjustment of the buffer reduced co-elution of PL species detected by the CAD with the result that the combined protocol yielded greater PL recoveries than that previously published, 2.30 ± 0.03 % total PL in milk. Efficiency and improved recoveries was also reflected in PL values obtained from different dairy streams, 35.32 ± 0.01 % total PL and 46.09 ± 0.01% total PL in buttermilk and butter serum, respectively. Most notably recoveries of the more acidic PLs, phosphatidylinositol (PI) and phosphatidylserine (PS) increased by 2 % and 7 % across all dairy streams analysed compared to previous studies. Generation of a novel dairy ingredient enriched in dairy PLs was achieved through development of a process at laboratory scale that combined enzymatic digestion and ultrafiltration (UF). Buttermilk with its higher residue of PL arising from buttermaking was prepared by reconstitution of buttermilk powder (BMP) substrate and subjected to extensive hydrolysis of its inherent milk protein complement followed by ultrafiltration to permeate the resulting smaller molecular weight peptide material. Screening of a number of digestive enzymes based on their proteolytic activity identified Alcalase® as the most proteolytic enzyme that generated a hydrolysate with 89.84 % of the peptides < 50 kDa in size. Comparison of 100 kDa and 50 kDa molecular weight cut off (MWCO) membranes (Sartorius Vivaflow™ 200 PES crossflow cassette) resulted in no significant difference in the peptide profiles of the obtained retentates. A 7.8-fold increase in PL (based on 6.16 ± 0.02 % and 0.79 ± 0.01 % total PL in the freeze-dried retentate and BMP starting material, respectively) was achieved based on degree of hydrolysis (DH) of 19 % and 50 kDa MWCO UF membrane filtration. This combined approach increased the lipid material 6.3-fold and reduced residual protein content 2-fold in the retentate compared to the starting BMP with no evidence of PLs detectable in the permeate, 0.00 ± 0.01 % total PL. Successful scalability of this combined process was achieved at pilot scale with an even higher 8.5-fold increase in PL material in the retentate compared to the starting material, 11.05 ± 0.02 % and 1.30 ± 0.00 % total PL, respectively. A total lipid increase of 8.7-fold and total residual protein decrease of 2.9-fold during scale-up in the pilot plant exceeded laboratory scale performance. Subsequent application of supercritical fluid extraction (SFE) treatment to the obtained PL enriched spray dried retentate yielded an enriched PL extract, 56.24 ± 0.07 % total PL (dry matter) depleted of protein and lactose, thus exceeding the dairy PL enrichment values achieved by previous researchers. Application of the highly-enriched PL extract in vitro determined a neurotrophic bioactivity. The PL extract induced a stimulatory effect on the outgrowth of cortical neuron cells. Much of the research published to date has explored the biological effects of long-chain polyunsaturated fatty acids (LC-PUFAs) strongly associated with PLs, e.g. docosahexaenoic acid (DHA). Investigation of the effect of the PL extract at different dosages determined that at a concentration of 150 µg mL-1 PL extract a 43 % increase in cortical neuron stimulation was observed compared to the control, 0 % stimulation. However, at a concentration of 300 µg mL-1 PL extract the observed stimulation decreased to 12 % suggesting a neurotoxic effect at higher dosage levels. This increase is greater than that previously published and the effective concentration, 150 µg mL-1, is in agreement with the PL levels found in human milk. This study demonstrated that dairy PLs promote the development and outgrowth of cortical neurons and open up an avenue to explore further the putative health benefits associated with dairy PLs.

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