Anatomy and Neuroscience - Doctoral Theses

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Adolescence as a vulnerable period for the effects of intrinsic and extrinsic regulators of neurogenesis on cognitive behaviour
(University College Cork, 2017) O'Leary, James Daniel; Nolan, Yvonne M.; Cryan, John F.; O'Leary, Olivia; Science Foundation Ireland
Postnatal hippocampal neurogenesis is the birth of new neurons within the dentate gyrus that occurs throughout the lifespan. In adulthood, these new neurons have been shown to be necessary for cognitive tasks such as spatial and contextual memory. It is well established that adult hippocampal neurogenesis can be modulated by a number of intrinsic and extrinsic factors, such as intracellular signalling molecules, exercise, inflammation and stress. Moreover, levels of adult hippocampal neurogenesis do not remain constant throughout life. Indeed, levels of hippocampal neurogenesis and integration of new neurons within the dentate gyrus are up to four times higher during adolescence than during adulthood. The first aim of this thesis (addressed in Chapter 2) was to explore the extent and involvement of Tlx in motor, cognitive and anxietyrelated behaviour. A spontaneous deletion of Tlx, a key intrinsic regulator of neurogenesis, was demonstrated to impair motor, cognitive and anxiety-related behaviours during adolescence and adulthood. The second aim of this thesis (addressed in Chapters 3 and 4) was to investigate the impact of adolescent-initiated exercise on hippocampal plasticity and contextual and cued fear conditioning as well as pattern separation in adulthood. It was demonstrated that adult-initiated exercise enhanced both contextual and cued fear conditioning, while conversely, exercise that began in adolescence did not affect performance in these tasks and these differential effects were accompanied by differential expression of plasticity-related genes in the hippocampus in adulthood. Moreover, adult and adolescent-initiated exercise enhanced cognitive flexibility and dendritic complexity of immature neurons in the dentate gyrus. The third aim of this thesis (addressed in Chapter 5) was to examine the impact of chronically elevated IL-1β on adult hippocampal neurogenesis and pattern separation. It was shown that chronic lentiviral-mediated overexpression of IL-1β within the dorsal hippocampus impaired neurogenesis and performance in its associated cognition, while sparing neurogenesis independent cognition. Finally, the fourth aim of this thesis (addressed in Chapter 6) was to explore the impact of chronic IL-1β, chronic unpredictable stress exposure, or a combination of an initial chronic IL-1β insult was examined following exposure to chronic unpredictable stress on learning and memory and depressive-like behaviours. It was shown that exposure to chronically elevated IL1β and chronic stress independently impair certain types of learning and memory and increased depressive-like behaviour. However, exposure to a sequential ‘two-hit’ of chronically elevated hippocampal IL-1β and chronic stress did not produce an exacerbated phenotype. In summary (Chapter 7), disruption of intrinsic regulators of neurogenesis, such as Tlx, or exposure to extrinsic factors, such as exercise or adverse stimuli, like inflammation and stress, and the consequent effect on cognition may provide insight into why adolescence is a vital period for correct conditioning of hippocampal function in later life.
Assessment of a novel computer aided learning tool in neuroanatomy education
(University College Cork, 2018) Javaid, Muhammad Asim; Toulouse, André; Cryan, John F.; Schellekens, Harriët
Impaired understanding of intricate neuroanatomical concepts and structural inter-relationships has been associated with a fear of managing neurology patients, called neurophobia, among medical trainees. As technology advances, the role of e-learning pedagogies becomes more important to supplement the traditional dissection / prosection and lecture-based pedagogies for teaching neuroanatomy to undergraduate students. However, despite the availability of a myriad of e-learning resources, the neuro (-anatomy-) phobia – neurophobia nexus prevails. The focus of the PhD was to investigate the difficulties associated with learning neuroanatomy and to develop and assess the efficacy of a novel e-learning tool for teaching neuroanatomy, in the context of the strengths and pitfalls of the currently available e-learning resources. Firstly, we sought to provide direct evidence of the medical and health science students’ perception regarding specific challenges associated with learning neuroanatomy. The initial results showed that neuroanatomy is perceived as a more difficult subject compared to other anatomy topics, with spinal pathways being the most challenging to learn. Participants believed that computer assisted learning and online resources could enhance neuroanatomy understanding and decrease their neurophobia. Next, in the context of the significance of e-learning for supplementing traditional pedagogies, we identified features of neuroanatomy web-resources that were valued by students and educators with regards to learning neuroanatomy of the spinal pathways. Participants identified strengths and weaknesses of existing neuroanatomy web-resources and ranked one resource above the others in terms of information delivery and integration of clinical, physiological and medical imaging correlates. This provides a novel user perspective on the influence of specific elements of neuroanatomy web-resources to improve instructional design and enhance learner performance. Finally, considering the data acquired from students and educators, a novel, interactive, neuroanatomy learning e-resource was developed to support teaching of the neuroanatomy of the spinal pathways. The instructional design included a discussion of the clinical interpretation of basic neuroanatomical facts to aid in neurological localization. The e-learning tool was assessed and evaluated by undergraduate medical and neuroscience students using neuroanatomy knowledge quizzes and Likert-scale perception questionnaires and compared to the previously identified best-ranked neuroanatomy e-resource. Participants’ opinion regarding the usefulness of various components of the tools was also gauged. The results showed that usage of the UCC e-resource led to a significant increase in participants’ knowledge of the neuroanatomy of the spinal pathways compared to students’ who did not use e-resources. Moreover, the participants reported a greater interest in learning neuroanatomy with the novel tool, showing a greater appreciation for it while learning clinical neurological correlates compared to those using the best available e-resource identified earlier. In summary, the prevailing problem of neurophobia could be addressed by enhancing student-interest. Technological e-learning pedagogies, with intelligently designed interactive user-interface and clinical correlation of basic neuroanatomical facts can play a pivotal role in helping students learn neuroanatomy and breaking the nexus between neuro (-anatomy-) phobia and neurophobia.
Bugs, breathing and blood pressure: the microbiota-gut-brain axis in cardiorespiratory control
(University College Cork, 2019) O'Connor, Karen M.; O'Halloran, Ken D.; Cryan, John; Department of Physiology, University College Cork; Science Foundation Ireland
Dysregulated microbiota-gut-brain axis communication adversely influences neurocontrol systems, consequently affecting brain behaviours. It is plausible that microbiota-gut-brain axis signalling has a role in the control of breathing and cardiovascular function, as networks that govern cardiorespiratory control reside within the brainstem, a region innervated by the vagus nerve, a key signalling pathway of the microbiota-gut-brain axis. Cardiovascular and respiratory diseases are serious, potentially life-threatening conditions with limited treatment options. Thus, improved understanding of the underlying pathophysiology and novel therapeutic approaches are required. We performed an assessment of cardiorespiratory physiology in animal models of modified gut microbiota [antibiotic-treated (ABX) and faecal microbiota transfer (FMT)], and sleep-disordered breathing [chronic intermittent hypoxia (CIH)-exposed guinea-pigs and rats]. We investigated if dietary prebiotic supplementation prevented CIH-induced cardiorespiratory dysfunction in rats. Whole-body plethysmography was used to record ventilation and metabolism in unanaesthetised animals during normoxia and chemostimulation. Under anaesthesia, cardiorespiratory assessments were performed during normoxia, chemosensory stimulation and drug administration. Brainstem neurochemistry was assessed by high-performance liquid chromatography. 16S rRNA and whole-metagenome shotgun sequencing was used to characterise the gut microbiota. ABX and FMT disrupted the gut microbiota, brain neurochemistry and intestinal integrity, blunting chemoreflex control of breathing. Decreased brainstem noradrenaline and altered gut microbiota as well as impaired respiratory and autonomic control were evident in CIH-exposed guinea-pigs. CIH–exposed rats developed cardiorespiratory pathologies and decreased gut Lactobacillus rhamnosus relative abundance. Prebiotic administration increased short-chain fatty acid concentrations, measured by gas chromatography, but Lactobacillus rhamnosus and cardiorespiratory dysfunctions were not restored. Several commensal and pathogenic bacterial species correlated with blood pressure parameters. Our findings add to emerging research exploring microbiota-gut-brain signalling in homeostatic systems, extending investigations to cardiorespiratory control. Our studies draw focus to the potential application of manipulation of the gut microbiota as an adjunctive therapy for cardiorespiratory disease.
Characterisation of the molecular mechanisms of dopaminergic axonal growth and their impairment in Parkinson's disease
(University College Cork, 2021) Anantha, Jayanth; O'Keeffe, Gerard W.; Sullivan, Aideen M.; Science Foundation Ireland
Parkinson’s disease (PD) is a neurodegenerative disease that affects 6.1 million people globally as of 2016. Axonal degeneration has been identified as the earliest event in the pathogenesis of the disease. For this reason, the application of neurotrophic growth factors for the protection and/or restoration of dopaminergic neurons and their axons has been the subject of intensive research efforts. Glial derived neurotrophic factor (GDNF) was the first of these growth factors to be tested. Despite an extensive body of pre-clinical evidence, clinical trials of GDNF did not meet their primary end points. Thus, the search for new neurotrophic growth factors that can promote the survival of dopaminergic neurons and restore axonal degeneration has remained an ongoing pursuit. Growth differentiation factor (GDF)5 is one such neurotrophic factor under consideration for PD therapy. Therefore, in this body of work, we evaluate the molecular mediators through which GDF5 promotes neurite growth in cellular models of PD. GDF5 is known to mediate its neurotrophic growth factor effects by regulating the BMP-Smad signalling pathway. Therefore, for GDF5 to be a suitable candidate therapy for PD, it is important that the cellular response to GDF5 is unaffected by the disease process. In Chapter 2, we demonstrate that GDF5 signalling is normal in cellular models of PD. Leucine rich repeat kinase (LRRK)2, both wild type and the PD associated G2019S mutant, had no impact on the BMP-Smad mediated transcriptional response thus, confirming the integrity of this pathway in the LRRK2 models of PD. In contrast, α-synuclein impaired the BMP-Smad transcriptional response in cells overexpressing α-synuclein. α-synuclein impaired both BMPR1B- and Smad1- mediated transcription, however, GDF5 and BMP2 treatment restored α-synuclein-mediated impairments in the BMP-Smad transcriptional response. Histone deacetylase (HDAC)5 also regulates the BMP-Smad pathway, and here we demonstrate that HDAC5 nuclear localization or sequestration is central to the regulation of the BMP-Smad pathway. Finally, we demonstrate that GDNF, unlike GDF5, cannot activate its downstream transcriptional response in the presence of α-synuclein. Taken together, these findings suggest that the GDF5-mediated transcriptional response is unimpaired in cellular models of PD. Despite various studies demonstrating the neurite growth promoting effects of GDF5, in vitro and in vivo, its downstream effectors have remained largely unknown. In Chapter 3, we use a proteomics-based approach to identify the global change in proteome and identify upregulated proteins which play a critical role in mediating the axonal and neurite growth effects of GDF5. We identify two proteins called serine threonine receptor associated protein (STRAP) and nucleoside diphosphate kinase (NME)1 as proteins that play a critical role in GDF5-mediated axonal growth in SH-SY5Y cells. Using siRNA-mediated gene silencing, we demonstrated that the expression of both STRAP and NME1 proteins is crucial for GDF5-mediated neurite growth. Furthermore, we report that the overexpression of STRAP or NME1 was sufficient to promote neurite growth in SH-SY5Y cells. Additionally, we also demonstrate that treatment with recombinant NME1 promoted neurite growth in dopaminergic neurons from the embryonic day (E) 14 rat ventral mesencephalon (VM). Finally, we show that the coexpression pattern of the STRAP and NME1, with midbrain dopaminergic neuronal markers was reduced in the PD brain. Since we establish the role of NME1 in promoting neurite growth in neuronal cells and dopaminergic neurons, we next examine the effects of NME1 in cellular models of PD in Chapter 4. We demonstrate that NME1 protects against 6 hydroxydopamine-induced neurite degeneration in SH-SY5Y cells and E14 rat VM dopaminergic neurons. Additionally, we also demonstrate that NME1 prevented α-synuclein-induced reductions in neurite growth in SH-SY5Y cells. This effect of NME1 was confirmed in cultured primary dopaminergic neurons of E14 VM transduced with AAV-α-synuclein viral vectors. NME1 also rescued neurite growth impairment in cells stably expressing the LRRK2 G2019S mutant. Cumulatively, these findings demonstrated that NME1 treatment can protect against neurite growth impairments in multiple models of PD. We also demonstrated that NME1 exerts its neurite growth-promoting effects through the ROR2 and RORα receptors, and the expression of these receptors is critical for the neurite growth promoting effects of NME1. In the human substantia nigra, we found that NME1 is co-expressed with a distinct set of genes that regulate mitochondrial respiration. Therefore, we finally explore the effects of NME1 on mitochondrial respiration in SH-SY5Y cells. We found that NME1 treatment increases maximal respiration and proton leak in SH-SY5Y cells which confirmed that NME1 regulates mitochondrial respiration and promotes neurite growth. In Chapter 5, we explore the immediate effectors of GDF5 on neuronal cells using a proteomics approach. Our proteomics screen revealed that GDF5 upregulated 14 proteins which had significant coexpression pattern with BMPR2 and multiple dopaminergic neuronal markers. The gene ontology analysis on the proteins upregulated by GDF5 revealed their involvement in oxidative phosphorylation and mitochondrial respiratory processes. We next performed a Seahorse mitochondrial respiration assay to study the effect of GDF5 on mitochondrial function in SH-SY5Y cells. This revealed that GDF5 lowered oxygen consumption rates in SH-SY5Y cells and reduced basal and maximal respiration. Additionally, we demonstrate that in cell lines stably expressing LRRK2 wild type or the G2019S mutant, the expression of BMP-Smad signalling proteins was unaltered. We demonstrated that GDF5 promotes neurite growth in these cells and induces a BMP-Smad dependent transcriptional response. In G2019S-LRRK2 cells, GDF5 lowered basal respiration, maximal respiration, and ATP production, thus lowering bioenergetic demand which is a characteristic of differentiated cells. In summary, the work presented in this thesis identifies new downstream effectors and effects of GDF5. We demonstrate that the GDF5-mediated regulation of BMP-Smad pathway is unimpaired in cellular models of PD. Additionally, the results emanating from this body of work also identify NME1 as a new growth factor with potential neuroprotective and restorative properties. Finally, we demonstrated that GDF5 regulates mitochondrial respiration by promoting differentiation in neuronal cells and that GDF5 rescues neurite growth impairments in cells stably expressing G2019S-LRRK2. Therefore, we conclude that GDF5 and NME1 are, and remain, candidate neurotrophic factors for neuroprotection in PD.
Characterisation of the role of canonical BMP-Smad 1/5/8 signalling in the development of ventral midbrain dopaminergic neurons
(University College Cork, 2013) Hegarty, Shane V.; Sullivan, Aideen M.; O'Keeffe, Gerard W.; Irish Research Council for Science Engineering and Technology
Ventral midbrain (VM) dopaminergic (DA) neurons, which project to the dorsal striatum via the nigrostriatal pathway, are progressively degenerated in Parkinson’s disease (PD). The identification of the instructive factors that regulate midbrain DA neuron development, and the subsequent elucidation of the molecular bases of their effects, is vital. Such an understanding would facilitate the generation of transplantable DA neurons from stem cells and the identification of developmentally-relevant neurotrophic factors, the two most promising therapeutic approaches for PD. Two related members of the bone morphogenetic protein (BMP) family, BMP2 and growth/differentiation factor (GDF) 5, which signal via a canonical Smad 1/5/8 signalling pathway, have been shown to have neurotrophic effects on midbrain DA neurons both in vitro and in vivo, and may function to regulate VM DA neuronal development. However, the molecular (signalling pathway(s)) and cellular (direct neuronal or indirect via glial cells) mechanisms of their effects remain to be elucidated. The present thesis hypothesised that canonical Smad signalling mediates the direct effects of BMP2 and GDF5 on the development of VM DA neurons. By activating, modulating and/or inhibiting various components of the BMP-Smad signalling pathway, this research demonstrated that GDF5- and BMP2-induced neurite outgrowth from midbrain DA neurons is dependent on BMP type I receptor activation of the Smad signalling pathway. The role of glial cell-line derived neurotrophic factor (GDNF)-signalling, dynamin-dependent endocytosis and Smad interacting protein-1 (Sip1) regulation, in the neurotrophic effects of BMP2 and GDF5 were determined. Finally, the in vitro development of VM neural stem cells (NSCs) was characterised, and the ability of GDF5 and BMP2 to induce these VM NSCs towards DA neuronal differentiation was investigated. Taken together, these experiments identify GDF5 and BMP2 as novel regulators of midbrain DA neuronal induction and differentiation, and demonstrate that their effects on DA neurons are mediated by canonical BMPR-Smad signalling.
Comparative compositional analysis of the gut microbiome in animal models of addiction and stress
(University College Cork, 2018) Peterson, Veronica L.; Cryan, John F.; Science Foundation Ireland
Intro: The microbiome-gut-brain axis (MGBA) has been shown to be instrumental to brain and behaviour, including psychological disorders affecting stress, depression, and anxiety. Many of the aforementioned disorders are comorbid with substance abuse disorders, and on a larger scale with addiction. In this body of work, we seek to characterize addiction-related phenotypes in the microbiome and correlate with measures of addiction-related behaviours, along with changes in the virome following chronic social stress. Aims: We first examined if vapor administration of ethanol is capable of altering the microbiome and to assess if substances of abuse, such as ethanol, can alter the microbiome outside of oral/gastrointestinal administration. To characterize addictive phenotypes behavioural measures of impulsivity, reward learning, and dopaminergic response to novelty were used. Methods: All studies were carried out in rodents. Following behavioural testing, gut microbiota samples were analysed with 16S rRNA gene amplicon sequencing. In the chronic social stress study, the virome was also sequenced via shot-gun metagenomic sequencing of faecal filtrate. Correlations were performed between microbiome measures and biological measures of behaviour, cytokines, corticosterone, and dopamine mRNA expression. Results: Chapter 2) Investigation of microbiome and vapor alcohol administration in mice showed significant alterations in microbiome; Chapter 3) A pre-clinical model of alcohol-addiction showed no significant differences in the microbiome by grouped phenotype. However, low abundance genus-level bacteria correlated to striatal dopamine mRNA expression. Chapter 4) A large heterogenous study of male and female rats revealed that female microbiome composition is more associated to addictive measures. However, within both male and female operational taxonomic units (OTUs) were significantly correlated to impulsivity measures. Chapter 5) Chronic social stress in male mice significantly affected bacterial and viral 1-14 composition. Viral richness was increased alongside decreases in bacterial richness. Discussion: Results from this body of work show that the microbiome may be impacted by addiction and psychosocial stress. Our findings suggest that there are subtle yet significant differences in microbiome composition between rats with different behavioural phenotypes. These findings indicate that associations to addictive-related behaviour occur in low-abundance (<5%) bacteria and are sex-specific. Furthermore, microbiome (bacteriome and virome) is impacted top-down, brain to gut via the hypothalamic-pituitary-adrenal axis, during chronic psychosocial stress. These initial investigations into stress and gut bacteriophage (aka phage) indicate that increases in phage richness are associated to decreases in bacterial richness and alpha diversity during stress. These novel findings seed the foundation for new research investigating MGBA in stress, substance abuse, and addiction. Further research is necessary to elucidate the mechanisms involved in this cross-communication of the MGBA. New insights into these challenging disorders of addiction and stress may provide unique therapies targeting the gut microbiome.
Defining the potential of class-IIa histone deacetylases as a therapeutic target for Parkinson’s disease
(University College Cork, 2021-11-15) Mazzocchi, Martina; O'Keeffe, Gerard W.; Sullivan, Aideen M.; Collins, Louise; Irish Research Council; Science Foundation Ireland
Parkinson’s disease (PD) is a neurodegenerative disease characterized by early dopaminergic (DA) neuron degeneration, coupled with intracellular aggregation of α-synuclein (α-syn) in Lewy Bodies (LB). Given the lack of a disease-modifying therapy for PD, histone deacetylases (HDACs) have emerged as potential druggable therapeutic targets. The neuroprotective effects of pan-HDAC inhibitors (HDIs) and of some specific HDIs have been tested in both in vitro and in vivo models of PD, showing various outcomes, however which is the best class of HDACs to be targeted for neuroprotection in PD is unclear. In the first experimental chapter, we used gene co-expression analysis to show that HDAC5 and HDAC9 were positively co-expressed with the DA neuronal markers TH, GIRK2 and ALDH1A1 in the human substantia nigra (SN). We then demonstrated that both siRNA against HDAC5 and HDAC9, and the HDI MC1568 which inhibits both HDAC5 and HDAC9, promoted neurite outgrowth in SH-SY5Y cells and DA neurons. Neurite growth was used as a single cell readout of neurotrophic action. Furthermore, MC1568 treatment increased the expression of the neurotrophic factor BMP2, and of its downstream effector SMAD1. A reporter assay revealed that these HDAC5 and HDAC9 siRNAs, or treatment with MC1568, increased BMP-Smad dependent transcription. Furthermore, inhibition of BMP signalling either pharmacologically with dorsomorphin, or by overexpression of Smad4 dominant negative or inhibitory Smad7, abolished the neurite outgrowth-promoting effects of BMP2. Moreover, siRNAs against HDAC5 or HDAC9, but not against HDAC4 or HDAC7, promoted neurite growth in the presence of WT and A53T α-syn. Treatment with MC1568 protected DA neurons from MPP+-induced neurodegeneration in vitro. These findings confirmed HDAC5 and HDAC9 as novel regulators of BMP-Smad neurotrophic factor signalling pathway, making them potential therapeutic targets for PD. In the second experimental chapter, we expanded our gene co-expression analysis and found that HDAC3, HDAC5, HDAC6 and HDAC9 were co-expressed with the DA neuronal markers SLC6A3 and NR4A2 in the human SN. A RT-qPCR confirmed that mRNA of all four HDACs exhibited similar temporal expression profiles during DA neuronal development. We next investigated whether class-specific pharmacological inhibition of these four HDACs could promote neurite growth. We found that inhibition of HDAC1 and HDAC3, or of HDAC3 alone, using RGFP109 or RGFP966 respectively, and inhibition of HDAC6 using ACY1215, did not promote neurite growth or affect survival in SH-SY5Y cells. Conversely, LMK235, which is a class-IIa-specific HDI which mainly inhibits HDAC4 and HDAC5, significantly increased both histone acetylation levels and neurite outgrowth in SH-SY5Y cells. Using a GFP reporter assay, we confirmed that administration of LMK235 increased Smad-dependent transcription. We then showed that inhibition of BMPR1B receptor in both SH-SY5Y cells and primary DA neurons prevented the neurite growth-promoting effects of LMK235. Furthermore, LMK235 was neuroprotective against MPP+ neurotoxic insult, and against wild type (WT) and A53T α-syn overexpression, in both SH-SY5Y cells and DA neurons. These data confirmed the neuroprotective effects of class-IIa HDAC inhibition in vitro. In the third experimental chapter, we investigated the cellular localisation of HDAC5 and the therapeutic potential of the classIIa inhibitor MC1568 in vivo, in 6-hydroxydopamine (6-OHDA) treated in vitro models of PD. We found that 6-OHDA treatment of both SH-SY5Y cells and DA neurons led to nuclear accumulation of HDAC5. This was prevented by PMA, which activates the canonical PKC pathway that is known to cause HDAC5 shuttling out of the nucleus. Having confirmed that MC1568 treatment protected SH-SY5Y cells from 6-OHDA-induced neurodegenration in vitro, we next examined whether peripheral administration (7 daily i.p. injections) of MC1568 was neuroprotective in the intrastriatal 6-OHDA lesion rat model of PD in vivo. Behavioural analysis at 8 and 12 days after lesion surgery showed that MC1568 treatment partially ameliorated 6-OHDA-induced forelimb akinesia. Post-mortem analysis of nigrostriatal integrity showed that MC1568 partially protected striatal dopaminergic terminals and DA neurons in the SN and prevented the increase in microglial numbers in both the striatum (ST) and SN. Finally, we confirmed that 6-OHDA lesion induced HDAC5 nuclear accumulation in DA neurons in the SN, which was prevented by MC1568 treatment. Collectively, these data rationalise the strategy of peripheral administration of MC1568 for neuroprotection in PD. In the fourth and final experimental chapter, we sought to determine whether HDAC5 nuclear accummulation occurred in other in vitro and in vivo models of PD . We verified that there are increased nuclear levels of HDAC5 in SH-SY5Y cells and in DA neurons treated with the neurotoxin MPP+, or in those overexpressing of α-syn or LRRK2 G2019S. We found that combined overexpression of nuclear-restricted HDAC5 and α-syn further increased neurite degeneration. Furthermore, activation of the canonical CaMK/PKC pathway prevented HDAC5 nuclear accumulation and the consequent neurite degeneration. Finally, we demonstrated that HDAC5 nuclear accumulation occurred in DA neurons in the SN, in both the MPTP mouse model and the AAV-αSyn rat model of PD. Collectively, the data presented in this thesis rationalise the future development of strategies focused on HDAC5 inhibition as a potential neuroprotective strategy for PD.
Differential roles of specific sub-regions of the longitudinal axis of the hippocampus in the behavioural and neurogenesis responses to stress and antidepressant drugs
(University College Cork, 2018) Rocha Levone, Brunno; O'Leary, Olivia; Cryan, John F.
Accumulating evidence suggests that the hippocampus is functionally segregated along its longitudinal axis into a dorsal (dHi) and a ventral sub-region (vHi). Indeed, recent gene expression studies suggest that the hippocampus has a gradient of gene expression and that the area between the dHi and vHi, the intermediate hippocampus (iHi), may also be functionally independent, but it remains understudied. The hippocampus is also one of few brain regions where neurogenesis, the birth of new neurons, occurs throughout life and this process has been shown to play roles in learning and memory as well as in responses to stress and antidepressants. These diverse roles may be related to the functional segregation of the hippocampus along its longitudinal axis. Indeed, the dorsal hippocampus (dHi) plays a predominant role in spatial learning and memory, while the ventral hippocampus (vHi) is predominantly involved in the regulation of anxiety, a behaviour impacted by stress and chronic treatment with some antidepressants. In vivo studies have shown that chronic stress and chronic antidepressant treatment change neurogenesis preferentially in the vHi rather than the dHi. However, whether these findings are due to differential intrinsic sensitivities of neural progenitor cells (NPCs) resident in the dHi, iHi or vHi in response to the stress hormone corticosterone or in response to antidepressants is unknown. Moreover, the roles of the dHi, iHi and vHi in the behavioural responses to chronic stress, a risk factor for depression and anxiety disorders, and in the behavioural responses to acute and chronic antidepressant treatment have not yet been investigated. Thus, the aims of this thesis were to determine whether NPCs isolated from the dHi, iHi and vHi have differential intrinsic sensitivities in response to the stress hormone corticosterone, the glucocorticoid receptor agonist dexamethasone, and the antidepressant, fluoxetine. To this end, we isolated NPCs from the three hippocampal sub-regions and cultured them for 4 h or 4 days in vitro with either corticosterone, fluoxetine, or corticosterone with fluoxetine or for 7 days in vitro with either corticosterone or dexamethasone. Cell proliferation, neuronal differentiation and maturation, nuclear GR expression and cell viability were then assessed. Moreover, we also aimed to determine the roles of each hippocampal sub-region on emotional behaviours and neuroendocrine response under baseline conditions, chronic psychosocial stress conditions and under chronic antidepressant treatment conditions. To this end, we performed stereotaxic surgeries in C57BL/6 mice to lesion the dHi, iHi or vHi with ibotenic acid. After recovery, animals were submitted to emotional behaviour and neuroendocrine tests under baseline conditions, or after chronic psychosocial stress, or after acute and chronic fluoxetine treatment. As result, we determined for the first time that NPCs isolated from the iHi and especially vHi are more sensitive to the effects of long-term exposure (7 DIV) to corticosterone and dexamethasone. Long-term (7 DIV) corticosterone and dexamethasone exposure also reduced nuclear GR expression preferentially in cells from the vHi. Fluoxetine alone did not have any effect on cell proliferation or neuronal differentiation or maturation. However, fluoxetine prevented corticosterone-induced reductions in neuronal differentiation after 4 DIV treatment and these effects were observed predominantly in the iHi and vHi cell cultures. In vivo experiments showed that vHi lesions reduced anxiety under baseline conditions. Under chronic psychosocial stress conditions, iHi lesions increased stress-induced social avoidance and the lesion of all sub-regions prevented chronic stress-induced anxiety; dHi and vHi lesions prevented stress-induced anhedonia and only vHi lesions caused antidepressant-like behaviour in the forced swim test and promoted active coping behaviour. In the antidepressant experiment, vHi lesions prevented the antidepressant effects of acute fluoxetine treatment while iHi lesions prevented the antidepressant effects of chronic fluoxetine treatment, and both iHi and vHi lesions prevented the anxiolytic effects of chronic fluoxetine treatment. Taken together, these findings show for the first time that iHi and vHi NPCs have increased intrinsic sensitivity to longer term exposure to the stress hormone corticosterone, and that the vHi seems to be an important sub-region for antidepressant-like effects under chronic stress. Also, both the iHi and vHi but not the dHi seem to modulate the antidepressant and anxiolytic effects of fluoxetine.
Effects of psychotropic drugs on the microbiota-gut-liver-brain axis
(University College Cork, 2019) Cussotto, Sofia; Cryan, John; Dinan, Timothy G.
There is a growing recognition of the involvement of the gut microbiota in drug metabolism and vice versa the impact of drug intake on the microbiome. In this thesis, we focus our attention on psychotropic medications (from the Greek root psychè = mind and tropòs = turning). With few isolated studies showing that brain-targeting medications can have antimicrobial activity in vitro, we sought to investigate the impact of psychotropics on the microbiome and intestinal physiology in vivo. Across a range of psychotropic medications, lithium, valproate, aripiprazole and fluoxetine significantly impacted the microbiome composition and diversity. These effects were not directly linked to changes in intestinal permeability or short-chain fatty acids levels (Chapter 2). The mood stabilisers lithium and valproate significantly impacted bile acid metabolism and targeted a set of bile-metabolising bacteria. Two mechanisms hypothesised as possible players in the bile-targeted effects of lithium and valproate, hepatic inflammation and intestinal permeability, did not seem to play any overt role in the disruption of bile pathways (Chapter 3). We next investigated whether perturbations of the microbiome, through administration of probiotics or antibiotics, could alter the pharmacokinetics of olanzapine and risperidone, two antipsychotic medications. Antibiotics increased the blood levels of olanzapine (AUC, area under the curve) but did not influence the absorption of risperidone. The antibiotics did not have a direct effect on the expression of CYPs involved in the metabolism of antipsychotics. Among the bacterial genera detected by 16S sequencing, the relative abundance of Alistipes significantly correlated with the AUC of olanzapine but not risperidone, suggesting that this bacterium might play a role in the pharamacokinetic alterations observed in olanzapine-treated rats (Chapter 4). Lastly, intrigued by the findings of Chapter 2, we moved on to look at the microbiome-targeting effects of psychotropic drugs in a human population, the Dutch LifeLines DEEP cohort. Although the small sample size and certain limitations which should be addressed in future population-based studies, minor effects of drug consumption on the human gut microbiota were detected (Chapter 5). Overall, these results provide novel insight on the role exerted by psychotropic medications on the microbiota-gut-liver-brain axis. Possible implications of this work include optimisation of drug efficacy or toxicity, use of the microbiome as a tool to distinguish responders from non-responders and improvement of personalised medicine.
Enduring neurobehavioural effects induced by early-life microbiota-gut-brain axis alterations
(University College Cork, 2018) Hecke Morais, Livia; Cryan, John F.; Dinan, Timothy G.; Science Foundation Ireland; APC Microbiome Institute, College of Medicine and Health, University College Cork; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
There is a growing appreciation of the importance of the bidirectional communication between our gut and brain on regulating the function and development of multiple physiological systems, including the central nervous system. Recently, the gut microbiota was demonstrated to interact with the gut-brain axis to regulate behaviour which has driven a paradigm shift in our understanding of neuropsychiatric disorders. An individual’s microbiota starts to develop mainly upon birth and continues to change throughout life. This initial colonization has a significant impact on development and maturation of the immune system. Conversely, disruptions of early-life microbiota have been implicated to long-lasting effects on stress, social behaviour and anxiety. Understanding the importance of early-life for shaping the gut-brain axis will further contribute to better strategies for disease prevention and treatment. This thesis investigated the impact of the gut-brain axis disruptions in early-life and the neurobehavioural consequences in two different scenarios: birth by C-section and maternal immune activation with polyinosinic-polycytidylic acid during pregnancy. To this end, here we developed a mouse model of C-section in NIH Swiss mice and demonstrated for the first time that the mode of delivery at birth can alter the stress-response, social behaviour, anxiety-like behaviour and cognition across the lifespan. These neurobehavioural deficits were associated with marked changes in the gut microbiota composition and diversity in early-life and adolescence with special decrease in Bifidobacterium spp. Further, we demonstrated that some deficits in social behaviour and cognition induced by the mode of delivery are reversed by targeting the gut microbiota in early-life through prebiotic, probiotic treatment, microbial transfer, and by pharmacological treatment with oxytocin. Complementary to the findings in mice some aspects of stress-related behavioural and physiological changes were also observed in a cohort of young-adults individuals. To further interrogate the importance of early-life for priming the gut-brain axis function in a different animal model, we investigated whether maternal immune activation with polyinosinic-polycytidylic acid (poly I:C) at gestational day 12.5 could behavioural, physiological and molecular aspects relevant to neurodevelopmental disorders in offspring of an outbred (NIH Swiss) and an inbred (C57BL6/J) strain. By looking at these two different strains we were able investigate whether gene and environment can interact in the susceptibility to develop gut-brain axis phenotype. We demonstrated that these strains differ in anxiety and depression-like behaviours with the effects being 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: stress and gut permeability. Taken together, these data suggest that gut-brain axis alterations in early-life may underpin altered programming of the developing brain and behaviours. Moreover, genetic background is a critical factor in susceptibility to the gut-brain axis alterations in certain conditions. Further efforts into understanding the factors that contribute to the major role for the gut-brain axis on programing brain health in early life may allow the development of new treatment strategies.
Expression and function of the neurotrophic factors GDF5 and GDNF in the nigrostriatal system during development and in rat models of Parkinson's disease
(University College Cork, 2013) Gavin, Aisling M.; Sullivan, Aideen M.; O'Keeffe, Gerard W.
Growth/differentiation factor 5 (GDF5) and glial cell line-derived neurotrophic factor (GDNF) are neurotrophic factors that promote the survival of midbrain dopaminergic neurons in vitro and in vivo. Both factors have potent neurotrophic and neuroprotective effects in rat models of Parkinson's disease (PD), and may represent promising new therapies for PD. The aim of the present study was to investigate the endogenous expression and function of GDF5 and GDNF in the nigrostriatal dopaminergic system during development and in rat models of PD. Examination of the temporal expression patterns of endogenous GDF5, GDNF, and their respective receptors, in the developing and adult nigrostriatal dopaminergic system suggest that these factors play important roles in promoting the survival and maturation of midbrain dopaminergic neurons during the period of postnatal programmed cell death. The relative levels of GDF5 and GDNF mRNAs in the midbrain and striatum, and their individual temporal expression patterns during development, suggest that their modes of actions are quite distinct in vivo. Furthermore, the sustained expression of GDF5, GDNF, and their receptors into adulthood suggest roles for these factors in the continued support and maintenance of mature nigrostriatal dopaminergic neurons. The present study found that endogenous GDF5, GDNF, and their receptors are differentially expressed in two 6-hydroxydopamine-induced lesion adult rat models of PD. In both terminal and axonal lesion models of PD, GDF5 mRNA levels in the striatum increased at 10 days post-lesion, while GDNF mRNA levels in the nigrostriatal system decreased at 10 and 28 days post-lesion. Thus, despite the fact that exogenous GDF5 and GDNF have similar effects on midbrain dopaminergic neurons in vitro and in vivo, their endogenous responses to a neurotoxic injury are quite distinct. These results highlight the importance of studying the temporal dynamic changes in neurotrophic factor expression during development and in animal models of PD.
From neurons to neuroblastoma: dietary flavonoids as regulators of life, death & differentiation
(University College Cork, 2020-03-24) Alshangiti, Amnah M.; O'Keeffe, Gerard W.; Sullivan, Aideen M.; Hegarty, Shane; Government of Saudi Arabia
Neuroblastoma (NB) is an embryonal malignancy that arises from cells of sympathoadrenal lineage during the development of the nervous system. Patients classified as stage 4 with aggressive tumors are considered to belong to a high-risk group. MYCN-amplification is detected as a hallmark of NB in 20% of high-risk cases. Based on the International Neuroblastoma Risk Group (INRG), the relapse survival rate of high-risk patients with MYCN-amplification is only 4%. Despite extensive studies, metastasis and tumor relapse lead to debilitating morbidity and frequently death, in high-risk NB patients. Moreover, NB chemotherapy can result in very debilitating long-term sequelae. Errors in the molecular mechanisms that control the development of cell of the sympathetic lineage have been linked to NB development. One group of signals, known as the bone morphogenetic proteins (BMPs), plays an important role in sympathetic neuritogenesis, by signaling through bone morphogenetic protein receptor (BMPR)2 and either BMPR1A or BMPR1B. Alterations in BMPR2 expression have been reported in NB, but it is unknown if the expression of BMPR1A or BMPR1B is altered. The use of neurotrophic factors such as nerve growth factor (NGF) and growth differentiation factor (GDF)5 to promote cell differentiation has been reported in postnatal day (P)1 mouse NC cultures and MYCN-amplified NB SK-N-BE(2), respectively. GDF5 induces cell differentiation in NB cells through activation of BMP-Smad signaling. However, recombinant BMPs are expensive to produce and their clinical use is limited, due to several factors. Therefore, there is an urgent need to identify novel therapeutic agents that could induce cell differentiation in post-chemotherapy tissue with the minimum side effects. Recently, several screening studies have identified small molecules that modulate the BMP pathway. These include natural dietary compounds, including flavonoids and in particular the chalcone subfamily. Interestingly, flavonoids have potential anti-tumor properties with selective cytotoxicity to cancer cells. Additionally, chalcones, which are the most potent flavonoid subclass, have also demonstrated anti-tumor activity in some contexts. This thesis first investigated, in chapter 2, the association of distinct type1 BMPRs with different molecular pathways and survival outcomes in NB. Lower expression of BMPR2 and BMPR1B, and higher BMPR1A expression, were observed in stage 4 and in MYCN-amplified NB, and they were associated with poor survival. In contrast, higher expression of BMPR2 and BMPR1B were associated with better survival. Regarding MYCN status, the correlation between BMPR2 and BMPR1A was strengthened, while the correlation between BMPR2 and BMPR1B was lost, in MYCN-amplified NB. This study therefore showed that expression of distinct BMPRs is associated with different survival outcomes in NB. In chapter 3, the effects of low doses of the chalcones isoliquiritigenin (ISLQ) and 4-hydroxychalcone (4HC), small molecule activator of BMP-Smad signaling, on the morphological differentiation of NB cells, as well as the molecular mechanism of their action, was investigated. ISLQ and 4HC promoted neurite growth in NB cells by activation of BMP-Smad signaling. Moreover, treatment with these chalcones lead to a significant reduction of cell aggregates (tumor-like aggregations) in SK-N-BE(2) cells. After these findings, in chapter 4, the cytotoxic effect of ISLQ in MYCN-amplified NB cells was investigated in detail. ISLQ induced cell death in MYCN-amplified NB cells SK-N-BE(2) and IMR-32 through an elevation of oxidative stress and necroptosis. Combined treatment of ISLQ with the chemotherapy agent cisplatin led to a greater cell toxicity in MYCN-amplified NB cells. In chapter 5, the cytotoxic effect of 4HC on NB cells, and its mode of action, was examined. 4HC demonstrated selective cytotoxicity on MYCN-amplified NB cells, which were more sensitive to 4HC than non-amplified SH-SY5Y cells. Cell death induced by 4HC was shown to result from glutathione (GSH) depletion, elevation of oxidative stress and mitochondrial impairment. This thesis, therefore, demonstrates the role of various types of BMPRs in different molecular pathways and survival outcomes in NB, It also highlights the potential of dietary chalcones for either the induction of NB cell differentiation at lower concentrations, through the activation of BMP-Smad signaling, or induction of selective NB cell death at higher concentrations, by elevation of oxidative stress.
Ghrelin system signalling in appetite and reward: in vitro and in vivo perspectives
(University College Cork, 2018) Howick, Kenneth P.; Griffin, Brendan T.; Schellekens, Harriët; FHI 360
The regulation of food intake is one of the most intricate internal balances in mammalian behaviour. Dysregulation of the central mechanisms underlying appetite control and metabolism result in both disorders of under- and over-eating. Disorders of appetite result in significant morbidity and mortality, and represent a major unmet clinical need. The endogenous hormone ghrelin and its receptor, the growth hormone secretagogue receptor (GHSR-1a), have long been known as pharmacological targets for appetite-related and metabolic disorders. Nutraceutical and bioactive peptides offer the opportunity to prevent onset and escalation of lifestyle-associated diseases of appetite and metabolism. However, there is a dearth of clinical evidence to justify the development of many bioactives as nutraceuticals. The potential applicability of dairy-derived bioactives in appetite-related disorders is now becoming increasingly apparent. We investigate whether a dairy-derived hydrolysate can increase GHSR-1a signalling in vitro, and whether this can be translated to evidence of effect in vivo in a pre-clinical model (Chapter 2). Subsequently, by leveraging advanced pharmaceutical technology, we develop a gastro-protective and sustained delivery system with a high payload capacity (Chapter 3). Furthermore, ligand-dependent biased signalling, and ligand biodistribution may have important roles to play in increasing efficacy of ghrelin ligands in vivo. Therefore, we investigate whether two synthetic ghrelin ligands, anamorelin and HM01, exert differential effects on the GHSR-1a in vitro (Chapter 4). The divergent effects of these two ligands on appetite and reward-motivated behaviours, as well as effects on central neuronal activation and reward system dopamine (DA) levels will also be investigated with a view to informing strategies to optimize future ghrelin therapies (Chapter 4 and 5). Chapter 2 and 3 provide an effective platform for gastro-protected delivery of bioactive peptides to enable further proof-of-concept studies across the appetite modulation field. Evidence of an orexigenic effect of the bioactive is seen in vivo in a rodent model. The oral delivery system developed served as a clinical formulation platform for proof-of-concept studies in humans to be conducted within the wider Food for Health Ireland research consortium. Chapters 4 and 5 show the importance of biased signalling and biodistribution of ghrelin ligands. Greater maximal food intake is reported by the brain penetrant HM01 vs. the peripherally limited anamorelin. Divergent neuronal activation of the two ligands is also shown in reward processing areas using c-fos immunostaining. Targeting specific downstream signalling pathways will enable the provision of more efficacious appetite modulation therapies, while centrally penetrant ligands will provide further therapeutic avenues through greater reward system activation.
The glutamatergic system and pain: influence of stress, oestrous cycle and gut microbiota
(University College Cork, 2019) Sajjad, Jahangir; O'Mahony, Siobhain M.; Cryan, John F.; University College Cork
Women have a higher incidence of various visceral and neuropathic chronic pain conditions. Most of the animal studies investigating pain mechanisms show oestrogen to be pronociceptive. However, similar studies in healthy humans are equivocal. The predisposition of chronic pain conditions in female patients with a history of stress emphasises the role of chronic stress in the pathogenesis of these conditions. Since glutamatergic system plays an import role in the transmission of pain-related information in the central nervous system (CNS), its impaired modulation may be a key phenomenon responsible for the pathogenesis of pain conditions and associated gender differences. The microbiota-gut-brain axis is shown to be capable of influencing the CNS function in health and various diseases, but as yet it is to be determined whether it has any role in the modulation of pain-related central nervous mechanisms. Extensive research is being carried out to understand the mechanisms through which sex hormones and chronic stress may influence the pain neurotransmission resulting in sex differences in pain. However, these mechanisms are not fully understood yet. As microbiota-gut-brain axis has been shown to influence a number of CNS disorders, and there is emerging evidence of its role in pain modulation in preclinical studies, we specifically concentrated on gut microbiota and its associated mechanisms influencing pain sensitivity. Our objectives were to explore the interplay between sex, stress, glutamatergic system, gut microbiota, and their effect on pain responses. We performed experiments both in animals and healthy humans. We were able to demonstrate that in rats, synaptic glutamate metabolism in the spinal cord and anterior cingulate cortex of brain varied significantly depending upon the phase of the oestrous cycle. Endogenous oestrogen exerted its effects through oestrogen receptor-α and was also shown to regulate glutamate receptor subunits expression. By utilising animal models of visceral hypersensitivity and stress (early-life stress & depression) in Wistar-Kyoto (WKY) and maternally separated rats, we could demonstrate that stress significantly altered pain responses through modifying the glutamatergic system and such effect was oestrous phase specific. In WKY rats, short-chain fatty acid producing gut microbiota abundance had a positive correlation with synaptic glutamate transporter function in a sex-specific manner. Finally, in healthy humans, we demonstrated a positive correlation between the abundance of butyrate producing bacteria in the bowel and pain sensitivity. Furthermore, hormonal contraceptive use was associated with increased lipopolysaccharide-binding protein levels. Taken together, our results show that oestrogen plays a vital role in glutamatergic neurotransmission and pain responses. Aberrant changes in the pain-related sensory mechanisms may develop by factors such as stress and altered gut microbiota. The emerging relationship between sex hormones, neuroendocrine system, and the gut microbiota in relation to pain modulation supports further investigations in large scale studies. Trials are needed to establish if interventions to correct gut microbiota imbalance can have analgesic effects.
Identifying novel molecular mechanisms of ghrelin receptor signalling underlying neural control of food intake: interaction with stress and impulsivity
(University College Cork, 2015) van Oeffelen, Wesley; Schellekens, Harriët; Cryan, John F.; Dinan, Timothy G.; Molecular Medicine Ireland
The gut-hormone, ghrelin, activates the centrally expressed growth hormone secretagogue 1a (GHS-R1a) receptor, or ghrelin receptor. The ghrelin receptor is a G-protein coupled receptor (GPCR) expressed in several brain regions, including the arcuate nucleus (Arc), lateral hypothalamus (LH), ventral tegmental area (VTA), nucleus accumbens (NAcc) and amygdala. Activation of the GHS-R1a mediates a multitude of biological activities, including release of growth hormone and food intake. The ghrelin signalling system also plays a key role in the hedonic aspects of food intake and activates the dopaminergic mesolimbic circuit involved in reward signalling. Recently, ghrelin has been shown to be involved in mediating a stress response and to mediate stress-induced food reward behaviour via its interaction with the HPA-axis at the level of the anterior pituitary. Here, we focus on the role of the GHS-R1a receptor in reward behaviour, including the motivation to eat, its anxiogenic effects, and its role in impulsive behaviour. We investigate the functional selectivity and pharmacology of GHS-R1a receptor ligands as well as crosstalk of the GHS-R1a receptor with the serotonin 2C (5-HT2C) receptor, which represent another major target in the regulation of eating behaviour, stress-sensitivity and impulse control disorders. We demonstrate, to our knowledge for the first time, the direct impact of GHS-R1a signalling on impulsive responding in a 2-choice serial reaction time task (2CSRTT) and show a role for the 5-HT2C receptor in modulating amphetamine-associated impulsive action. Finally, we investigate differential gene expression patterns in the mesocorticolimbic pathway, specifically in the NAcc and PFC, between innate low- and high-impulsive rats. Together, these findings are poised to have important implications in the development of novel treatment strategies to combat eating disorders, including obesity and binge eating disorders as well as impulse control disorders, including, substance abuse and addiction, attention deficit hyperactivity disorder (ADHD) and mood disorders.
The impact of maternal inflammation and maternal stress in the regulation of neurodevelopment and physiological function
(University College Cork, 2014) Crampton, Seán; O'Keeffe, Gerard
The mechanisms governing fetal development follow a tightly regulated pattern of progression such that interference at any one particular stage is likely to have consequences for all other stages of development in the physiological system that has been affected thereafter. These disturbances can take the form of many different events but two of the most common and widely implicated in causing detrimental effects to the developing fetus are maternal immune activation (MIA) and maternal stress. MIA has been shown to cause an increase in circulating proinflammatory cytokines in both the maternal and fetal circulation. This increase in proinflammatory mediators in the fetus is thought to occur by fetal production rather than through exchange between the maternal-fetal interface. In the case of maternal stress it is increased levels of stress related hormones such as cortisol/corticosterone which is thought to elicit the detrimental effects on fetal development. In the case of both maternal infection and stress the timing and nature of the insult generally dictates the severity and type of effects seen in affected offspring. We investigated the effect of a proinflammatory environment on neural precursor cells of which exposure resulted in a significant decrease in the normal rate of proliferation of NPCs in culture but did not have any effect on cell survival. These effects were seen to be age dependent. Using a restraint stress model we investigated the effects of prenatal stress on the development of a number of different physiological systems in the same cohort of animals. PNS animals exhibited a number of aberrant changes in cardiovascular function with altered responses to stress and hypertension, modifications in respiratory responses to hypercapnic and hypoxic challenges and discrepancies in gastrointestinal innervation. Taken together these findings suggest that both maternal infection and maternal stress are detrimental to the normal development of the fetus.
Innovative strategies for teaching anatomy to dental students
(University College Cork, 2018) Lone, Mutahira; Downer, Eric J.; McKenna, Joseph P.; Toulouse, André; Cryan, John F.; University College Cork
Anatomy education is an integral component of the undergraduate and postgraduate dental curriculum. A detailed understanding of anatomy is a pre-requisite before examination, diagnosis and clinical treatment of patients in all aspects of the healthcare systems. Anatomy teaching is undergoing pioneering changes. Traditional Vesalius’ dissection-based teaching has evolved to include didactic lectures and nowadays incorporates digital teaching, e-learning and a wide range of 3D images and models. Furthermore, the current generation of dental students are using devices like smartphones and laptops for educational purposes. The focus of this PhD was to assess the efficacy of innovative teaching aids for teaching anatomy to dental students. We hypothesized that innovative tools are effective in enhancing students learning experience and improving academic performance. Firstly, a cranial nerve animation was developed and results show an increase in student’s knowledge of the topic after watching the animation (Chapter 2). The teaching of clinical skills was assessed using a soft-preserved cadaver (Chapter 3) which showed promising results. Furthermore, to aid in teaching tooth morphology a 3D quiz application was developed (Chapter 5) and results show that it was effective in teaching tooth morphology to dental students. Additionally, a survey was also undertaken to assess the staff involved in teaching tooth morphology to dental students in the UK and Ireland and the teaching aids employed for teaching and examination of tooth morphology (Chapter 4). Students’ feedback and perceptions were also gathered at the end of each study. The results show that innovative teaching aids can supplement the traditional teaching methods. Furthermore, students benefitted from using the innovative teaching aids and reported it as a positive learning experience. Hence, accepting the above stated alternate hypothesis that innovative tools were effective in enhancing students learning experience and improved their academic performance. In summary, the principles of anatomy are best taught through traditional teaching methods which are reinforced by the various innovative supplemental teaching aids available. A blended teaching model has been recommended with traditional methods complimented by innovative pedagogies. The introduction of these innovative technologies is vital in providing educational support to students and are successful when delivered alongside taught lectures and the use of the traditional teaching methods.
Investigating the developmental and behavioral consequences of maternal immune activation on affected offspring
(University College Cork, 2015) Straley, Megan E.; O'Keeffe, Gerard; Irish Research Council
Maternal infection during pregnancy increases the risk of several neuropsychiatric disorders later in life, many of which have a component of dopaminergic (DA) dysfunction, including schizophrenia, autism spectrum disorders (ASD), and attention deficit hyperactivity disorder (ADHD). The majority of DA neurons are found in the adult midbrain; as such the midbrain is a key region of interest regarding these disorders. The literature is conflicting regarding the behavioral alterations following maternal immune activation (MIA) exposure, and the cellular and molecular consequences of MIA on the developing midbrain remain to be fully elucidated. Thus, this thesis aimed to establish the consequences of acute and mild MIA on offspring dopamine-related behaviors, as well as the associated cellular and molecular disturbances of MIA on offspring midbrains. We utilized a rat model of MIA using low dose (50μg/kg, I.P.) of LPS administered at different gestational ages. Our first study indicated that MIA at later gestational ages significantly increased pro-inflammatory IL-1β expression, and reduced HSD11B2 expression in the placenta, which is an important regulator of fetal development. In utero LPS exposure at later gestational ages also impaired the growth of neurons from affected offspring. This study identified key gestational stages during which MIA resulted in differential effects. We utilized these time points in subsequent studies, the next of which investigated neurobehavioral outcomes following MIA. Our results from that study showed that motor differences occurred in juvenile offspring following MIA at E16 only, and these differences were compensated for in adolescence. Then, there was a decline in motor behavior capabilities in adulthood, again only for animals exposed to MIA on E16 (and not E12). Furthermore, our results also demonstrated adolescent and adult offspring that were exposed to MIA at E12 had diminished responses to amphetamine in reward seeking behaviors. In our final study, we aimed to investigate the molecular and cellular changes following MIA which might explain these behavioral alterations. This final study showed a differential inflammatory response in fetal midbrains depending on gestational age of exposure as well as differential developmental alterations. For example, LPS exposure at E16 resulted in decreased VM neurosphere size after 7DIV and this was associated with an increased susceptibility to neurotoxic effects of pro-inflammatory cytokines for VM neurospheres and VM DA neurons treated in culture. In utero LPS exposure at E16 also reduced DA neuron count of fetal VM, measured by TH staining. However, there were no differences in DA neuron number in juvenile, adolescent, or adult offspring. Similarly, LPS exposure did not alter cell number or morphology of glial cells in the midbrains of affected offspring. In conclusion, this thesis indicated later rat pregnancy (E16) as vulnerable time for MIA to affect the development of the nigrostriatal pathway and subsequent behavioral outcomes, possibly implicating a role for MIA in increased risk for disorders associated with motor behavior, like PD. These effects may be mediated through alterations in the placenta and altered inflammatory mediators in the offspring brain. This thesis has also shown that MIA in earlier rat pregnancy (E12) results in altered mesocorticolimbic function, and in particular MIA on E12 resulted in a differential response to amphetamine in affected offspring, which may implicate a role for MIA in increasing the risk for disorders associated with this pathway, including drug tolerance and addiction.
Investigating the mechanisms by which PINK1 protects against Parkinson's disease
(University College Cork, 2019) Furlong, Rachel; O'Neill, Cora; Sullivan, Aideen M.; Irish Research Council
PINK1 (PTEN-induced putative kinase-1) functions as a ubiquitous serine-threonine kinase, with pro-survival, neuroprotective and anti-stress signalling functions. Autosomal recessive mutations in PINK1, resulting in a loss of PINK1 function, cause early onset Parkinson’s disease (PD). This thesis aimed to investigate the mechanisms by which PINK1 protects against PD, with a focus on PI3-kinase/Akt signalling. Akt signalling is central to cell survival, metabolism, protein and lipid homeostasis, and is impaired in PD. Akt activation is reduced in the PD brain, and by many PD-causing genes, including PINK1. However, it is not yet known how PINK1 regulates PI3-kinase/Akt signalling. Chapter Two investigated the mechanisms by which PINK1 regulates Akt signalling in PINK1 modified mouse embryonic fibroblasts (MEFs). Our results reveal for the first time that PINK1 constitutively activates Akt in a PINK1-kinase dependent manner in the absence of growth factors and enhances Akt activation in normal growth medium. In PINK1 modified MEFs, agonist-induced Akt signalling failed in the absence of PINK1, due to significantly impaired PINK1 kinase-dependent increases in PI(3,4,5)P3 at both the plasma membrane and Golgi. In the absence of PINK1, PI(3,4,5)P3 levels did not increase in the Golgi, and there was significant Golgi fragmentation, a recognised characteristic of PD neuropathology. PINK1 kinase activity protected the Golgi from fragmentation in an Akt-dependent fashion. This demonstrates a new role for PINK1 as an upstream activator of Akt via PINK1 kinase-dependent regulation of its primary activator PI(3,4,5)P3, providing novel mechanistic information on how loss of PINK1 impairs Akt signalling in PD. Despite its discovery nearly two decades ago, there is a lack of cohesive information on PINK1 binding partners. Determining how PINK1 interacts with proteins involved in certain signalling pathways or cellular processes is crucial for understanding how loss of PINK1 function leads to PD. Chapter 3 aimed to assemble a comprehensive list of known PINK1- interacting proteins and to identify new candidates that are bound to PINK1, using bioinformatics analysis of existing data sets coupled with mass spectrometry approaches. This study identified PINK1-interacting proteins that are heavily involved in receptor tyrosine kinase (RTK) signalling pathways, such as PI3-kinase/Akt, VEGF, PDGF, ERRB2/4 and EGF, indicating that PINK1 plays a key functional role through these pathways. Further analysis highlighted two PINK1-interacting proteins (PTEN and IRS4) that are involved in PI3-kinase/Akt signalling and which may mediate PINK1’s regulation of this pathway through lipid binding with PIP3. Using mass spectrometry approaches, several new binding proteins for PINK1 were identified, including RIPK3, calmodulin, EGFR, and interferon regulatory genes, all of which have been implicated in PD and as components of the Akt signalling system. Accumulation of α-synuclein is central to the development of PD, and mutations in SNCA, which encodes α-synuclein, cause PD. There is a need to identify the mechanisms by which α-synuclein overexpression induces neurodegeneration in PD and to determine whether αsynuclein and PINK1 share common mechanisms. This information will be critical for future identification of therapeutic targets. In Chapter 4, we identified significant Golgi fragmentation, mitochondrial fission and reductions in neurite length in cultured ventral midbrain neurons, in response to overexpression of wildtype or mutant α-synuclein, and to deletion of PINK1. Increased Golgi fragmentation and mitochondrial fission induced by the PD risk genes were significantly correlated with reductions in neurite length. Levels of PI(3,4,5)P3 were significantly decreased by overexpression of wildtype or mutant αsynuclein. α-synuclein overexpression in combination with knockdown of PINK1 induced a “dual-hit”, to further reduce neurite length and increase Golgi fragmentation. This provides novel data showing that α-synuclein overexpression and PINK1 deletion converge to induce significant increases in Golgi fragmentation and mitochondrial fission, with concomitant decreases in neurite length and defects in PI3-kinase/Akt signalling. This thesis therefore demonstrates that studies on the function of PD-causing genes such as PINK1 and α-synuclein enable the discovery of molecular pathways that may be involved in the pathogenesis of PD and could be targets for future therapies.
Investigating the regulatory role of the nuclear receptor TLX in IL-1β-induced changes in hippocampal neurogenesis
(University College Cork, 2017) Ó Léime, Ciarán S.; Nolan, Yvonne M.; Cryan, John F.
Hippocampal neurogenesis is the process by which new neurons are born within the dentate gyrus (DG). This process begins during embryonic development and persists throughout life. Neurogenesis encompasses proliferation, differentiation and integration of neural progenitor cells (NPCs) into the surrounding neural network. Each stage is regulated by a host of intrinsic and extrinsic factor such as intracellular signalling molecules, exercise, environmental enrichment, diet and learning. TLX is an orphan nuclear receptor and transcription factor, which promotes the proliferation of NPCs, maintains the neurogenic pool of cells within the DG, and has been shown to promote hippocampal neurogenesis-associated cognition. Conversely, the proinflammatory cytokine IL-1β is a major mediator of the anti-neurogenic effects of hippocampal neuroinflammation, and previous work from the group has shown that IL-1β can suppress the expression of TLX within proliferating NPCs. The aims of this thesis were to investigate the interactions between TLX and IL-1β both in vitro and in vivo, and to determine the behavioural outcome of enhancing TLX and IL-1β, as well as well as in response to dietary intervention in vivo. We demonstrate that IL-1β suppresses TLX expression and neurogenesis (neurosphere expansion) in vitro, and that these effects are mediated by the NF-κB pathway. Restoration of TLX expression is sufficient to attenuate the negative effects of IL-1β on neurogenesis. We have shown using an RNA sequencing approach that TLX expression maintains a reduced inflammatory transcriptional profile in the hippocampus at baseline, and regulates the transcriptional response to IL-1β in vivo. We demonstrate that lentiviral-mediated overexpression of TLX does not enhance hippocampal neurogenesis-associated cognitive processes in vivo but that it impairs object recognition memory in rats. This suggests that enhancing cell proliferation is not sufficient to promote certain hippocampal-associated cognitive processes, and may even have a detrimental effect on cognitive behaviour. Finally, we show that an adolescent cafeteria diet which induces negative effects on hippocampal-associated memory, does not induce lasting cognitive defects when rats are switched to standard chow diet in adulthood. Lentiviral-mediated overexpression of IL-1β does not impact upon cognitive behaviours in rats fed a cafeteria diet throughout adolescence. However, we show that this chronic low-grade hippocampal IL-1β-mediated inflammation promotes fear memory in adulthood. In summary, TLX and IL-1β can enhance and repress hippocampal neurogenesis respectively. Determining the role that TLX has on neurogenesis-associated cognition and how it can interact with IL-1β may position TLX as a novel therapeutic target for the treatment of neuroinflammatory-associated disorders where hippocampal neurogenesis is impaired.

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