Anatomy and Neuroscience - Doctoral Theses

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    Peripheral alterations underlying the negative effects of a cafeteria diet on brain and behaviour: exercise as a mitigating strategy
    (University College Cork, 2024) Nota, Minke H. C.; Nolan, Yvonne M.; O'Leary, Olivia; Irish Research Council; Science Foundation Ireland
    A Western lifestyle, characterised by inactivity and overconsumption of saturated fats and sugar, increases risk of depression, anxiety, and cognitive impairment. Obesity, metabolic dysfunction, (neuro)inflammation, and gut microbiota alterations, which can result from a Western lifestyle, are associated with mood disorders and cognitive impairment, thus constituting potential mechanisms by which Western lifestyle impacts the brain. Adult hippocampal neurogenesis (AHN), i.e., the birth of new neurons in the dentate gyrus of the hippocampus, is involved in certain forms of memory, including spatial memory and pattern separation, and in regulating emotion through anxiety behaviours and antidepressant action. A cafeteria (CAF) diet, mimicking human Western-style diets, has been shown to decrease AHN, impair memory, and increase anxiety-like behaviour in rodents, whereas exercise has antidepressant effects and has been shown to improve AHN and cognition. However, interactions between these lifestyle factors remain unclear. Furthermore, effects of Western-style diets and exercise on AHN and associated behaviours have primarily been researched in males, whereas depression, certain anxiety disorders, and dementia disproportionally affect women. The aims of this thesis were to investigate whether voluntary running exercise could alter the effects of a CAF diet on AHN and hippocampus-associated behaviour, and the intake of and preference for a CAF diet in adult male and female rats, and to determine if a CAF diet and exercise could impact metabolic markers, inflammation, and gut-derived metabolites in males. Exercise had anxiolytic effects in males and females, induced modest improvements in spatial learning in males, and decreased spatial memory in females. Additionally, exercise mitigated a CAF diet-induced increase in depression-like behaviour, and a CAF diet blunted an exercise-induced increase in AHN, in males but not females. In exercising males and females with access to CAF diet, intake of energy from CAF foods and saturated fat was decreased, and fibre and protein intake was increased compared to sedentary rats with access to a CAF diet. Moreover, compared to sedentary rats, exercising rats had reduced preference for CAF foods over standard chow, which was maintained for 2 and 5 weeks in females and males, respectively. Increased hypothalamic Drd1 gene expression, which has been shown to promote overeating, in exercising males with access to a CAF diet possibly explained reduced preference for CAF foods in exercising rats not being maintained past 5 weeks. Alterations in metabolic hormones and caecal metabolites offer potential explanations for behavioural and neurogenic effects observed in males. Exercise-induced increased PYY potentially contributed to anxiolytic effects of exercise, and a CAF diet attenuating exercise-induced increased GLP-1 possibly explained the blunting of neurogenic effects of exercise. Attenuation of CAF diet-induced increased leptin and insulin and decreased caecal indole-3-carboxylate and deoxyinosine by exercise potentially contributed to exercise mitigating CAF diet-induced increased depression-like behaviour. Furthermore, exercise attenuated a CAF diet-induced decrease in abundance of caecal anserine, a metabolite previously associated with improved cognitive function. No definitive pro- or anti-inflammatory pattern of a CAF diet and exercise emerged that might have contributed to changes observed in behaviour and AHN. However, ventral hippocampal Il-6r gene expression was decreased in exercising males with access to a CAF diet, possibly explaining the finding that in males, exercise attenuated a CAF diet-induced increase in depression-like behaviour, as reduced general IL-6 activity has been associated with antidepressant effects. Ultimately, these data highlight the importance of exercise combined with a healthy diet for hippocampal health, along with sex differences in lifestyle influences on brain and behaviour. Moreover, these data indicate potential mechanisms, including metabolic hormones and gut microbial metabolites, underlying interactions between a CAF diet and exercise on brain and behaviour, thereby aiding advancement of preventative measures for depression and cognitive impairment.
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    Unravelling the role of circadian rhythmicity in microbiota-gut-brain axis signalling
    (University College Cork, 2023) Tofani Sousa e Silva, Gabriel; Cryan, John; Saks-Kavanaugh Foundation; Science Foundation Ireland
    Modern habits are becoming more and more disruptive to health. Our days are often filled with circadian disruption and stress exposure. We need to understand how our responses to these external stimuli are shaped and how they can be targeted to promote health. A growing body of research demonstrates the role of the gut microbiota in influencing brain function and behavior. The stress and circadian systems, which are essential to maintaining appropriate responses to the environment, are known to be impacted by the gut microbiota. Although gut microbes have been shown to modulate circadian rhythms and stress response, such studies were conducted in an independent manner. Since these systems are interconnected through the hypothalamic-pituitary-adrenal (HPA) axis, there is a need to examine how the gut microbiota may play a role in regulating the integration of stress and circadian signals. In this thesis, I aimed to investigate the influence of circadian rhythmicity in the microbiota-gut-brain axis communication and the consequences of that to stress responsivity. To this end we developed a computational tool, Kronos, that allows us to determine rhythmicity of genes and metabolites in brain regions important to stress and circadian regulation under different microbial status. Moreover, we investigate the interplay of the circadian and stress systems in the absence of the microbiota to dissect its role in the modulation of these systems. Using transcriptomics, and applying Kronos, we demonstrate that microbial depletion by antibiotic administration or germ-free status disrupts the molecular clock in the superchiasmatic nucleus (SCN). Such alterations to the master clock were accompanied with disruptions in the rhythmicity of circulating corticosterone. Furthermore, multi-omics analysis in the hippocampus and amygdala, indicated that microbial status disrupted the diurnal oscillations in genes and metabolites that compose pathways important for the stress response. We then investigate the expression of genes related to circadian rhythms and HPA-axis in the paraventricular nucleus, pituitary, and adrenal glands across the day. This, together with alterations in corticosterone demonstrates a hyper-activation of the HPA-axis at the sleep/wake transition in microbial depleted animals. Such disruption to the rhythmic function of the HPA-axis resulted in a time-of-day specific impairment of the stress response and stress-sensitive behaviors. Lastly, we identify changes in the rhythmic profile of the gut microbiota following microbial depletion. This manifested as peak of bacterial load at the same time the impairments in HPA-axis function were observed. Furthermore, by conducting fecal microbiota transplantation we confirm that the diurnal oscillations in gut microbes after antibiotic treatment regulates glucocorticoids secretion and explore the microbial compositional changes underlying it. This work provides compelling evidence that the gut microbiota regulates stress responsivity via the circadian system. Moreover, we identify the gut microbiota as an important regulator of HPA-axis rhythmic function, demonstrating that the microbiota is essential to adaptively respond to psychological stressors throughout the day.
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    The bacteriome and virome in social and stress-related disorders
    (University College Cork, 2023) Ritz, Nathaniel L.; Cryan, John; Dinan, Timothy G.; Science Foundation Ireland
    The gastrointestinal microbiota has been shown to influence mammalian health and well-being in many conditions but has also been implicated in a variety of diseases and disorders. The multi-faceted interaction of the microbiota with the host that affects the brain and behaviour is termed the microbiota-gut-brain axis. While this axis plays an important role in stress and social behaviour, research has largely focused on associations with the bacterial fraction of the community. The gut also harbours viruses, fungi, archaea, and other microbial forms of life that can affect the health of the mammalian host. Gut viruses, which are dominated by bacteriophages, are also ubiquitous in the gut and have the ability to infect bacteria and alter their structure and activity. Moreover, these populations of viruses are intimately associated with the bacterial community, but little is known about their involvement within the microbiota-gut-brain axis. Whether bacteriophages are capable of exerting effects on the brain and behaviour is largely unknown. To this end, we tested whether the virome was affected by stress followed by the capability of the virome to prevent stress-related sequelae. We found that the virome is stress sensitive and that restoring the virome to mice undergoing stress reduced anxiety-like behaviour, immune activation, inflammation, and gene expression in the brain. These data provide evidence that the gut virome can be harnessed to improve stress-coping outcomes. Social diseases and disorders have been implicated in the microbiota-gut-brain axis. Social anxiety disorder is a common but understudied psychiatric condition. Whether specific microbiota communities play a role in social fear responsivity or are capable of driving social fear behaviour is also unknown. Preclinical animal models of social fear allow for testing translational hypotheses that can then be returned to the clinic. We sought to characterize associations of the microbiota with social fear resiliency and susceptibility. Then, we transferred microbiota from individuals with social anxiety disorder to mice to test whether the microbiota played a causative role in the disorder. We found a strong correlation between microbiota and social fear variability along with differential expression of amygdalar genes involved in social behaviour, immunity, and host-microbe interaction. We also found that microbiota from individuals with social anxiety disorder could drive social fear sensitivity in mice. Mice that received microbiota from social anxiety disorder donors also had impaired cytokine release in the gut, reduced immune cell populations in the mesenteric lymph nodes and circulation, altered basal HPA axis, and diminished oxytocin and oxytocin-related expression of genes in the brain. These data indicate that social fear and anxiety are linked with the microbiota and that the gut-brain axis is a target for future research for social anxiety disorder therapeutics. Herein we tested whether gut viruses could alter stress response, whether social fear behaviour is associated with differential microbial communities, and whether human microbiota from individuals with social anxiety disorder can drive social fear behaviour in the mouse. Collectively we have shown that stress-related sequelae can be restored by virome transfer and that the microbiota can play a causal role in social anxiety disorder.
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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.
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    Defining the potential of ZNHIT1, an SNCA co-expressed gene in the substantia nigra, as a therapeutic target for Parkinson’s disease
    (University College Cork, 2022) McCarthy, Erin; O'Keeffe, Gerard W.; Sullivan, Aideen M.; Collins, Louise; Irish Research Council
    Parkinson’s Disease (PD) is synucleinopathy that is characterised by the formation of toxic α-Synuclein (αSyn)-containing Lewy Bodies (LBs) in the midbrain leading to the progressing death of dopaminergic (DAergic) neurons in the substantia nigra (SN). Toxic aggregation of αSyn results in the dysfunction of important neuronal processes, leading to increased neurotoxicity and neurodegeneration. SNCA and its mutant variants have been linked to several cases of familial PD. Given the lack of effective disease-modifying therapies, there is an increasing focus on examining SNCA-induced changes in epigenetic regulation in the hopes of identifying novel targets for gene therapy in PD. In the first experimental chapter, we used gene co-expression analysis to identify Synuclein Alpha (SNCA) co-expressed genes in the SN, whose co-expression pattern was lost in PD. We identified nuclear zinc finger HIT-type containing 1 (ZNHIT1) as an important interacting partner of SNCA in the SN, and that this co-expression pattern is lost in PD indicating functional dysregulation.. We went on to investigate the functional role of ZNHIT1, which revealed that overexpression of ZNHIT1 promotes neurite growth and prevents αSyn-induced reductions in neurite growth and cell viability in SH-SY5Y cells. Analysis of ZNHIT1 co-expressed genes in the SN revealed a significant enrichment of genes associated with the regulation of mitochondrial function. Bioenergetic state analysis agreed with these findings and revealed that ZNHIT1 overexpression increases ATP synthesis, and rescues αSyn-induced impairments in oxygen consumption rate (OCR), basal respiration, maximal respiration, and spare respiratory capacity. These findings reveal that ZNHIT1 can protect against αSyn-induced neurotoxicity and mitochondrial dysfunction in ZNHIT1-overexpressing cells, this rationalising further investigation into ZNHIT1 as a potential therapeutic target for PD. In the second experimental chapter, we investigated the role of ZNHIT1 in αSyn-induced neurotoxicity and mitochondrial dysfunction in PD. PD is characterised by impairments in mitochondrial function and reductions in ATP levels. ZNHIT1 overexpression protects against αSyn-induced deficits in mitochondrial function through an upregulation of genes associated with mitochondrial function. Proteomic and bioinformatic analysis revealed that ZNHIT1 interacts with mitochondrial proteins that are significantly enriched in functional categories important for mitochondrial function such as mitochondrial transport, ATP synthesis, and ATP-dependent activity. We also found that ZNHIT1 upregulates and is co-expressed with hub protein HSP90B1, which is known to deter PD progression, thus indicating a neuroprotective role for ZNHIT1-HSP90BI in the SN. Indeed, we show that ZNHIT1 is also co-expressed with DAergic markers TH and ALDH1A1 in control samples, but that this correlation is lost in PD samples. These results indicate functional dysregulation of ZNHIT1 in PD that may result in the misregulation of its mitochondrial interacting proteins in the cytosol, leading to mitochondrial dysfunction and reductions in ATP synthesis that is characteristic of PD, and thus validates our previous findings that highlight ZNHIT1 as a potential target for PD therapy In the third experimental chapter, we investigated the role of ZNHIT1 in BMP-Smad-dependent transcriptional activation in SH-SY5Y cells overexpressing ZNHIT1. Our analysis revealed that ZNHIT1 activates the BMP-Smad pathway, which has been shown to promote DAergic neurite growth and survival and protects them against αSyn-induced neurotoxicity. However, SNCA overexpression was found to inhibit these ZNHIT1-induced increases in BMP-Smad activation. Further investigation revealed that the neuroprotective effects of ZNHIT1 against αSyn-induced cellular and mitochondrial dysfunction, were inhibited by the BMP receptor (BMPR) inhibitors, Dorsomorphin and K02288, indicating that the neuroprotective effects of ZNHIT1 may be dependent on BMP-Smad signalling. We also show that SMAD4 expression in SH-SY5Y cells overexpressing dominant negative SMAD4 was rescued by ZNHIT1 overexpressing. These results support the hypothesis that αSyn in PD inhibits BMP-Smad signalling, which could lead to the inhibition of the growth promoting effects of ZNHIT1, which appear to be mediated by BMP-Smad signalling. Together, these results further highlight the potential role of ZNHIT1 as a therapeutic target for PD. In the fourth and final experimental chapter, we sought to examine the gene expression changes associated with important signalling networks induced by αSyn overexpression in an in vivo AAV-αSyn rat model of PD, in order to further our understanding of the role of αSyn in PD pathology. Analysis of gene expression changes of 84 genes known to be associated with PD pathology revealed significant reductions in the expression of genes associated with DA synthesis. Further analysis of gene expression changes in the SN induced by αSyn revealed 2,305 differentially expressed genes. The top ranked gene to be overexpressed in this list was Skor1, a known inhibitor of BMP-Smad signalling. Further investigation revealed a significant reduction in constitutively active BMPR1B-stimulated luciferase activity in HEK293T and SH-SY5Y cells overexpressing αSyn. Gene set enrichment analysis (GSEA) revealed that the overexpression of αSyn causes disruptions to cytoskeletal organisation, DNA repair networks and ATP binding, while analysis of cellular bioenergetic states showed reduced ATP synthesis, oxygen consumption rates and basal rates of respiration. This study highlights the role of αSyn as a regulator of mitochondrial function, ATP synthesis and BMP-Smad signalling. Collectively, the data presented in this thesis rationalises the future development of strategies focused on ZNHIT1 overexpression as a potential neuroprotective strategy for PD. In this thesis, we show that overexpression of ZNHIT1 is neuroprotective against αSyn-induced neurotoxicity in PD, including reductions in cell viability and growth, as well as mitochondrial dysfunction, ATP synthesis and BMP-Smad signalling. We also show a functional dysregulation of ZNHIT1-SNCA in PD, suggesting that altered expression patterns of ZNHIT1 may play an important role in PD progression. We hypothesis that overexpression of ZNHIT1 in the SN of PD patients may result in neuroprotection against the progression of PD. Our results highlight a potential role for ZNHIT1 in cellular dysfunction that may underlie PD pathology. Collectively the data in this thesis rationalises the future development of strategies focused on ZNHIT1 overexpression as a potential neuroprotective strategy for PD.