Anatomy and Neuroscience - Doctoral Theseshttps://hdl.handle.net/10468/5602024-03-28T12:27:01Z2024-03-28T12:27:01Z471Adolescence as a vulnerable period for the effects of intrinsic and extrinsic regulators of neurogenesis on cognitive behaviourO'Leary, James Danielhttps://hdl.handle.net/10468/61002023-04-04T07:16:46Z2017-01-01T00:00:00Zdc.title: Adolescence as a vulnerable period for the effects of intrinsic and extrinsic regulators of neurogenesis on cognitive behaviour
dc.contributor.author: O'Leary, James Daniel
dc.description.abstract: 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.
2017-01-01T00:00:00ZAn investigation into mechanisms of visceral pain in rodentsFelice, Valeria D.https://hdl.handle.net/10468/18582023-04-04T07:19:56Z2014-01-01T00:00:00Zdc.title: An investigation into mechanisms of visceral pain in rodents
dc.contributor.author: Felice, Valeria D.
dc.description.abstract: Visceral pain is a debilitating symptom of irritable bowel syndrome (IBS), a disorder affecting up to 30% of adults. A better understanding of the mechanisms underlying visceral hypersensitivity may facilitate development of more targeted therapies, improving the quality of life of these individuals. The studies performed in this thesis were designed to investigate important factors of visceral pain, including early-life manipulations, genetic predisposition and sex hormones. Maternal separation (MS) consistently reproduces visceral hypersensitivity and altered anxiety-like behaviours in rats, symptoms associated with IBS. It has been found that 5-HT2B receptor antagonism blocks visceral pain but no difference in relative 5-HT2B receptor mRNA expression was found in hippocampus, amygdala and colon. The neuronal activation patterns of prefrontal cortex and amygdala of MS rats were then investigated. MS animals are characterised by differential activation of the prefrontal cortex (anterior cingulate cortex (ACC), infralibic cortex, prelimbic cortex) as well as the central nucleus of the amygdala (CeA). Genetic factors also contribute to pain syndromes such as IBS. We utilised the Wistar Kyoto (WKY) rat, a stress-sensitive strain, as an animal model of brain-gut axis dysfunction. WKY rats have a lower expression of the glutamate transporter EAAT2 and mGlu4 receptor in the ACC. Another early-life factor that can increase susceptibility to functional gastrointestinal symptoms later life is disruption of the gut microbiota, thus early-life antibiotic treatment was used to assess this effect. Antibiotic treatment induced visceral hypersensitivity in adulthood and may be related to observed reductions in spinal cord alpha-2A adrenoreceptor (adra2A) mRNA. Lastly, we investigated sex differences in visceral sensitivity. EAAT1 & 2 mRNA levels are lower in females, potentially increasing glutamatergic concentration at the symaptic level. Moreover, NR1 and NR2B subunits mRNA of NMDA receptor were increased in caudal ACC of females. These findings may account for sex differences in visceral sensitivity.
2014-01-01T00:00:00ZAssessment of a novel computer aided learning tool in neuroanatomy educationJavaid, Muhammad Asimhttps://hdl.handle.net/10468/83652023-04-04T07:16:03Z2018-01-01T00:00:00Zdc.title: Assessment of a novel computer aided learning tool in neuroanatomy education
dc.contributor.author: Javaid, Muhammad Asim
dc.description.abstract: 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.
2018-01-01T00:00:00ZBugs, breathing and blood pressure: the microbiota-gut-brain axis in cardiorespiratory controlO'Connor, Karen M.https://hdl.handle.net/10468/94762023-04-04T07:14:09Z2019-01-01T00:00:00Zdc.title: Bugs, breathing and blood pressure: the microbiota-gut-brain axis in cardiorespiratory control
dc.contributor.author: O'Connor, Karen M.
dc.description.abstract: 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.
2019-01-01T00:00:00ZCharacterisation of the molecular mechanisms of dopaminergic axonal growth and their impairment in Parkinson's diseaseAnantha, Jayanthhttps://hdl.handle.net/10468/136462023-04-04T10:45:33Z2021-01-01T00:00:00Zdc.title: Characterisation of the molecular mechanisms of dopaminergic axonal growth and their impairment in Parkinson's disease
dc.contributor.author: Anantha, Jayanth
dc.description.abstract: 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.
2021-01-01T00:00:00ZCharacterisation of the role of canonical BMP-Smad 1/5/8 signalling in the development of ventral midbrain dopaminergic neuronsHegarty, Shane V.https://hdl.handle.net/10468/19732023-04-04T07:26:57Z2013-01-01T00:00:00Zdc.title: Characterisation of the role of canonical BMP-Smad 1/5/8 signalling in the development of ventral midbrain dopaminergic neurons
dc.contributor.author: Hegarty, Shane V.
dc.description.abstract: 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.
2013-01-01T00:00:00ZComparative compositional analysis of the gut microbiome in animal models of addiction and stressPeterson, Veronica L.https://hdl.handle.net/10468/79412023-04-04T07:41:01Z2018-01-01T00:00:00Zdc.title: Comparative compositional analysis of the gut microbiome in animal models of addiction and stress
dc.contributor.author: Peterson, Veronica L.
dc.description.abstract: 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.
2018-01-01T00:00:00ZDefining the potential of class-IIa histone deacetylases as a therapeutic target for Parkinson’s diseaseMazzocchi, Martinahttps://hdl.handle.net/10468/124942023-04-04T11:02:58Z2021-11-15T00:00:00Zdc.title: Defining the potential of class-IIa histone deacetylases as a therapeutic target for Parkinson’s disease
dc.contributor.author: Mazzocchi, Martina
dc.description.abstract: 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.
2021-11-15T00:00:00ZDefining the potential of ZNHIT1, an SNCA co-expressed gene in the substantia nigra, as a therapeutic target for Parkinson’s diseaseMcCarthy, Erinhttps://hdl.handle.net/10468/154722024-02-01T02:06:17Z2022-01-01T00:00:00Zdc.title: Defining the potential of ZNHIT1, an SNCA co-expressed gene in the substantia nigra, as a therapeutic target for Parkinson’s disease
dc.contributor.author: McCarthy, Erin
dc.description.abstract: 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.
2022-01-01T00:00:00ZDifferential roles of specific sub-regions of the longitudinal axis of the hippocampus in the behavioural and neurogenesis responses to stress and antidepressant drugsRocha Levone, Brunnohttps://hdl.handle.net/10468/67822023-04-04T07:15:23Z2018-01-01T00:00:00Zdc.title: Differential roles of specific sub-regions of the longitudinal axis of the hippocampus in the behavioural and neurogenesis responses to stress and antidepressant drugs
dc.contributor.author: Rocha Levone, Brunno
dc.description.abstract: 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.
2018-01-01T00:00:00Z