Anatomy and Neuroscience - Doctoral Theses

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The role of the dopaminergic neurotrophin growth/differentiation factor-5 in the developing rat ventral midbrain
(University College Cork, 2004-10) O'Keeffe, Gerard W.; Sullivan, Aideen M.; Irish Research Council for Science Engineering and Technology
Growth differentiation factor-5 (GDF-5) is a member of the transforming growth factor-β superfamily, a family of proteins that play diverse roles in many aspects of cell growth, proliferation and differentiation. GDF-5 has also been shown to be a trophic factor for embryonic midbrain dopaminergic neurons in vitro (Krieglstein et al. 1995) and after transplantation to adult rats in vivo (Sullivan et al. 1998). GDF-5 has also been shown to have neuroprotective and neurorestorative effects on adult dopaminergic neurons in the substantia nigra in animal models of Parkinson’s disease (Sullivan et al. 1997, 1999; Hurley et al. 2004). This experimental evidence has lead to GDF-5 being proposed as a neurotrophic factor with potential for use in the treatment of Parkinson’s disease. However, it is not know if GDF-5 is expressed in the brain and whether it plays a role in dopaminergic neuron development. The experiments presented here aim to address these questions. To that end this thesis is divided into five separate studies each addressing a particular question associated with GDF-5 and its expression patterns and roles during the development of the rat midbrain. Expression of the GDF-5 in the developing rat ventral mesencephalon (VM) was found to begin at E12 and peak on E14, the day that dopaminergic neurons undergo terminal differentiation. In the adult rat, GDF-5 was found to be restricted to heart and brain, being expressed in many areas of the brain, including striatum and midbrain. This indicated a role for GDF-5 in the development and maintenance of dopaminergic neurons. The appropriate receptors for GDF-5 (BMPR-II and BMPR-Ib) were found to be expressed at high levels in the rat VM at E14 and BMPR-II expression was demonstrated on dopaminergic neurons in the E13 mouse VM. GDF-5 resulted in a three-fold increase in the numbers of dopaminergic neurons in cultures of E14 rat VM, without affecting the numbers of neurones or total cells. GDF-5 was found to increase the proportion of neurons that were dopaminergic. The numbers of Nurr1-positive cells were not affected by GDF-5 treatment, but GDF-5 did increase the numbers of Nurr1- positive cells that expressed tyrosine hydroxylase (TH). Taken together this data indicated that GDF-5 increases the conversion of Nurr1-positive, TH-negative cells to Nurr1-positive, TH-positive cells. In GDF-5 treated cultures, total neurite length, neurite arborisation and somal area of dopaminergic were all significantly increased compared to control cultures. Thus this study showed that GDF-5 increased the numbers and morphological differentiation of VM dopaminergic neurones in vitro. In order to examine if GDF-5 could induce a dopaminergic phenotype in neural progenitor cells, neurosphere cultures prepared from embryonic rat VM were established. The effect of the gestational age of the donor VM on the proportion of cell types generated from neurospheres from E12, E13 and E14 VM was examined. Dopaminergic neurons could only be generated from neurospheres which were prepared from E12 VM. Thus in subsequent studies the effect of GDF-5 on dopaminergic induction was examined in progentior cell cultures prepared from the E12 rat VM. In primary cultures of E12 rat VM, GDF-5 increased the numbers of TH-positive cells without affecting the proliferation or survival of these cells. In cultures of expanded neural progenitor cells from the E12 rat VM, GDF-5 increased the expression of Nurr1 and TH, an action that was dependent on signalling through the BMPR-Ib receptor. Taken together, these experiments provide evidence that GDF-5 is expressed in the developing rat VM, is involved in both the induction of a dopaminergic phenotype in cells of the VM and in the subsequent morphological development of these dopaminergic neurons
Novel insights into the inflammatory basis of gastrointestinal disease
(University College Cork, 2013) Moloney, Gerard M.; Nally, Kenneth; Cryan, John F.; Science Foundation Ireland
It has become clear that inflammation is beneficial to man, there are situations though that the inflammatory response causes damage to the host that is harmful to health. When the inflammatory response fails or is too strong, the health of the host is damaged and disease can occur. The implication of intestinal disease caused by an ineffective immune response is of great social and economic burden to society. The overarching purpose of this thesis is to assess inflammatory signalling targets associated with immune mediated disorders such as IBD, IBS and inflammatory liver disease. By assessing these targets and modifying their function I hope to contribute and expand further the pre-existing information on these disorders and improve the therapeutic interventions available in these debilitating conditions. I will assess the role of inflammation in disorders of the GI tract and liver IBD, IBS, hepatic inflammatory injury and furthermore, I will use pharmaceutical agents to activate and suppress components of the immune system. I will examine the inflammatory response in experimental models of disease for IBD and liver injury, I will attempt to alter these pathways using pharmaceutical intervention to delineate the disease causing mechanism that may lead to clinically relevant therapeutic interventions. In regards to IBS, I will attempt to improve the existing knowledge that exists in relation to the pathogenesis of this functional bowel disorder. I will attempt to define a mechanism by which the low grade mucosal inflammation that has been demonstrated by others arises and what this inflammation is induced by. The overall aim of this thesis is to attempt to further understand the mechanisms behind GI and liver disease. Looking at the inflammatory response in these specific conditions and how they can be altered may lead to exciting new therapies for inflammatory conditions in the gastrointestinal tract.
The role of glucocorticoid-induced tumour necrosis factor receptor in developing mouse sympathetic neurons
(University College Cork, 2013) McKelvey, Laura; O'Keeffe, Gerard W.; Science Foundation Ireland
Hereditary sensory autonomic neuropathy IV (HSAN IV) is an autosomal recessive disorder characterised by inability to feel pain and anhidrosis and is a consequence of defective NGF/TrkA signalling and growth of sensory and sympathetic neurons. Glucocortiocoid-induced tumour necrosis factors receptor (GITR), a transmembrane protein, activated by its specific ligand, GITRL, is well known for its role in the regulation of innate and acquired immune system responses. Recently, GITR was found to be required for NGF-dependant and extracellular signal-related kinase 1/2 (ERK1/2)-induced neurite growth and target innervation in the developing sympathetic nervous system (SNS). Given this novel role of GITR, it is possible that strategies targeting GITR have potential therapeutic benefit in promoting neurite growth in autonomic neuropathies such as HSAN IV. Using P1 mouse SCG neurons as a model, in addition to various SCG cell treatments, knock down models and transfection methods, we investigated whether GITR increases the sensitivity of sympathetic neurons to NGF; the region of GITR required for the enhancement of NGF-promoted growth, the signalling pathways downstream of GITR and how extensively GITR is involved in regulating peripheral innervation of the SNS. Results indicate that the region responsible for the growth promoting effects of GITR lies in its juxtamembrane intracellular region (here termed the growth promoting domain (GPD)) of GITR. The GPD of GITR activates ERK1/2 and inhibits nuclear factor kappa B (NF-κB) in an inverse fashion to provide an optimal cellular growth environment for P1 SCG neurons. While deleting the GPD of GITR had no effect on TrkA expression, constitutive phosphorylation of specific sites in the GPD reduced TrkA expression indicating a possible role for GITR in increasing the sensitivity of SCG neurons to NGF by the regulation of these sites, TrkA expression and subsequent NGF/TrkA binding. GITR appears to be heterogeneously required for NGF-promoted target innervation of SCG neurons in some organs, implying additional factors are involved in extensive NGF-target innervation of the SNS. In conclusion, this study answers basic biological questions regarding the molecular mechanism behind the role of GITR in the development of the SNS, and provides a basis for future research if GITR modulation is to be developed as a strategy for promoting axonal growth.
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.
The role of the IL-1 type 1 receptor in the life, death and differentiation of adult hippocampal neural precursor cells
(University College Cork, 2013) Ryan, Sinead Maria; Nolan, Yvonne M.; Science Foundation Ireland
Neurogenesis occurs in two distinct regions of the adult brain; the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus, and the subventricular zone (SVZ) lining the lateral ventricles. It is now well-known that adult hippocampal neurogenesis can be modulated by a number of intrinsic and extrinsic factors e.g. local signalling molecules, exercise, environmental enrichment and learning. Moreover, levels of adult hippocampal neurogenesis decrease with age, at least in rodents, and alterations in hippocampal neurogenesis have been reported in animal models and human studies of neuropsychiatric and neurodegenerative conditions. Neuroinflammation is a common pathological feature of these conditions and is also a potent modulator of adult hippocampal neurogenesis. Recently, the orphan nuclear receptor TLX has been identified as an important regulator of adult hippocampal neurogenesis as its expression is necessary to maintain the neural precursor cell (NPC) pool in the adult DG. Likewise, exposure of animals to voluntary exercise has been consistently demonstrated to promote adult hippocampal neurogenesis. Lentivirus (LV)- mediated gene transfer is a useful tool to elucidate gene function and to explore potential therapeutic candidates across an array of conditions as it facilitates sustained gene expression in both dividing and post-mitotic cell populations. Both intrinsic and extrinsic factors are important regulators of adult hippocampal neurogenesis. Examining how these factors are affected by an inflammatory stimulus, and the subsequent effects on adult hippocampal neurogenesis provides important information for the development of novel treatment strategies for neuropsychiatric and neurodegenerative conditions in which adult hippocampal neurogenesis is impaired. The aims of the series of experiments presented in this thesis were to examine the effect of the pro-inflammatory cytokine interleukin-1β (IL-1β) on adult hippocampal NPCs both in vitro and in vivo. In vitro, we have shown that IL-1β reduces proliferation of adult hippocampal NPCs in a dose and time-dependent manner. In addition, we have demonstrated that TLX expression is reduced by IL-1β. Blockade of IL-1β signalling prevented both the IL-1β-induced reduction in cell proliferation and TLX expression. In vivo, we examined the effect of short term and long term exposure to LV-IL-1β in sedentary mice and in mice exposed to voluntary running. We demonstrated that impaired hippocampal neurogenesis is only evident after long term exposure to IL-1β. In mice exposed to voluntary running, hippocampal neurogenesis is significantly increased following short-term but not long-term exposure to running. Moreover, short-term running effectively prevents any IL-1β-induced effects on hippocampal neurogenesis; however, no such effects are seen following long-term exposure to running.
The role of mitogen activated protein kinase phosphatase-1 in neurotoxic and inflammatory-induced changes in the development of midbrain dopaminergic neurons: relevance to Parkinson’s disease
(University College Cork, 2013) Collins, Louise M.; Nolan, Yvonne M.; Toulouse, André; College of Medicine and Health, University College Cork
Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterised by the loss of midbrain dopaminergic neurons from the substantia nigra pars compacta(SNpc), which results in motor, cognitive and psychiatric symptoms. Evidence supports a role for the mitogen-activated protein kinase p38 in the demise of dopaminergic neurons, while mitogen-activated protein kinase phosphatase-1 (MKP-1), which negatively regulates p38 activity, has not yet been investigated in this context. Inflammation may also be associated with the neuropathology of PD due to evidence of increased levels of proinflammatory cytokines such as interleukin-1β (IL-1β) within the SNpc. Because of the specific loss of dopaminergic neurons in a discreet region of the brain, PD is considered a suitable candidate for cell replacement therapy but challenges remain to optimise dopaminergic cell survival and morphological development. The present thesis examined the role of MKP-1 in neurotoxic and inflammatory-induced changes in the development of midbrain dopaminergic neurons. We show that MKP-1 is expressed in dopaminergic neurons cultured from embryonic day (E) 14 rat ventral mesencephalon (VM). Inhibition of dopaminergic neurite growth induced by treatment of rat VM neurons with the dopaminergic neurotoxin 6- hydroxydopamine (6-OHDA) is mediated by p38, and is concomitant with a significant and selective decrease in MKP-1 expression in these neurons. Dopaminergic neurons transfected to overexpress MKP-1 displayed a more complex morphology and contributed to neuroprotection against the effects of 6-OHDA. Therefore, MKP-1 expression can promote the growth and elaboration of dopaminergic neuronal processes and can help protect them from the neurotoxic effects of 6-OHDA. Neural precursor cells (NPCs) have emerged as promising alternative candidates to fetal VM for cell replacement strategies in PD. Here we show that phosphorylated (and thus activated) p38 and MKP-1 are expressed at basal levels in untreated E14 rat VM NPCs (nestin, DCX, GFAP and DAT-positive cells) following proliferation as well as in their differentiated progeny (DCX, DAT, GFAP and βIII-tubulin) in vitro. Challenge with 6-OHDA or IL-1β changed the expression of endogenous phospho-p38 and MKP-1 in these cells in a time-dependent manner, and so the dynamic balance in expression may mediate the detrimental effects of neurotoxicity and inflammation in proliferating and differentiating NPCs. We demonstrate that there was an up-regulation in MKP-1 mRNA expression in adult rat midbrain tissue 4 days post lesion in two rat models of PD; the 6-OHDA medial forebrain bundle (MFB) model and the four-site 6-OHDA striatal lesion model. This was concomitant with a decrease in tyrosine hydroxylase (TH) mRNA expression at 4 and 10 days post-lesion in the MFB model and 10 and 28 days post-lesion in the striatal lesion model. There was no change in mRNA expression of the pro-apoptotic gene, bax and the anti-apoptotic gene, bcl-2 in the midbrain and striatum. These data suggest that the early and transient upregulation of MKP-1 mRNA in the midbrain at 4 days post-6-OHDA administration may be indicative of an attempt by dopaminergic neurons in the midbrain to protect against the neurotoxic effects of 6-OHDA at later time points. Collectively, these findings show that MKP-1 is expressed by developing and adult dopaminergic neurons in the midbrain, and can promote their morphological development. MKP-1 also exerts neuroprotective effects against dopaminergic neurotoxins in vitro, and its expression in dopaminergic neurons can be modulated by inflammatory and neurotoxic insults both in vitro and in vivo. Thus, these data contribute to the information needed to develop therapeutic strategies for protecting midbrain dopaminergic neurons in the context of PD.
P-glycoprotein inhibition as a strategy to increase drug delivery across the blood-brain barrier: focus on antidepressants
(University College Cork, 2013) O'Brien, Fionn E.; Cryan, John F.; Griffin, Brendan T.
Depression is among the leading causes of disability worldwide. Currently available antidepressant drugs have unsatisfactory efficacy, with up to 60% of depressed patients failing to respond adequately to treatment. Emerging evidence has highlighted a potential role for the efflux transporter P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), in the aetiology of treatment-resistant depression. In this thesis, the potential of P-gp inhibition as a strategy to enhance the brain distribution and pharmacodynamic effects of antidepressant drugs was investigated. Pharmacokinetic studies demonstrated that administration of the P-gp inhibitors verapamil or cyclosporin A (CsA) enhanced the BBB transport of the antidepressants imipramine and escitalopram in vivo. Furthermore, both imipramine and escitalopram were identified as transported substrates of human P-gp in vitro. Contrastingly, human P-gp exerted no effect on the transport of four other antidepressants (amitriptyline, duloxetine, fluoxetine and mirtazapine) in vitro. Pharmacodynamic studies revealed that pre-treatment with verapamil augmented the behavioural effects of escitalopram in the tail suspension test (TST) of antidepressant-like activity in mice. Moreover, pre-treatment with CsA exacerbated the behavioural manifestation of an escitalopram-induced mouse model of serotonin syndrome, a serious adverse reaction associated with serotonergic drugs. This finding highlights the potential for unwanted side-effects which may occur due to increasing brain levels of antidepressants by P-gp inhibition, although further studies are needed to fully elucidate the mechanism(s) at play. Taken together, the research outlined in this thesis indicates that P-gp may restrict brain concentrations of escitalopram and imipramine in patients. Moreover, we show that increasing the brain distribution of an antidepressant by P-gp inhibition can result in an augmentation of antidepressant-like activity in vivo. These findings raise the possibility that P-gp inhibition may represent a potentially beneficial strategy to augment antidepressant treatment in clinical practice. Further studies are now warranted to evaluate the safety and efficacy of this approach.
Investigation of micro-devices for neurobiological applications
(University College Cork, 2013) Grygoryev, Konstantin; Galvin, Paul; McDermott, Kieran; Herzog, Grégoire; Arrigan, Damien; National Biophotonics and Imaging Platform Ireland; Higher Education Authority
The aim of this project is to integrate neuronal cell culture with commercial or in-house built micro-electrode arrays and MEMS devices. The resulting device is intended to support neuronal cell culture on its surface, expose specific portions of a neuronal population to different environments using microfluidic gradients and stimulate/record neuronal electrical activity using micro-electrode arrays. Additionally, through integration of chemical surface patterning, such device can be used to build neuronal cell networks of specific size, conformation and composition. The design of this device takes inspiration from the nervous system because its development and regeneration are heavily influenced by surface chemistry and fluidic gradients. Hence, this device is intended to be a step forward in neuroscience research because it utilizes similar concepts to those found in nature. The large part of this research revolved around solving technical issues associated with integration of biology, surface chemistry, electrophysiology and microfluidics. Commercially available microelectrode arrays (MEAs) are mechanically and chemically brittle making them unsuitable for certain surface modification and micro-fluidic integration techniques described in the literature. In order to successfully integrate all the aspects into one device, some techniques were heavily modified to ensure that their effects on MEA were minimal. In terms of experimental work, this thesis consists of 3 parts. The first part dealt with characterization and optimization of surface patterning and micro-fluidic perfusion. Through extensive image analysis, the optimal conditions required for micro-contact printing and micro-fluidic perfusion were determined. The second part used a number of optimized techniques and successfully applied these to culturing patterned neural cells on a range of substrates including: Pyrex, cyclo-olefin and SiN coated Pyrex. The second part also described culturing neurons on MEAs and recording electrophysiological activity. The third part of the thesis described integration of MEAs with patterned neuronal culture and microfluidic devices. Although integration of all methodologies proved difficult, a large amount of data relating to biocompatibility, neuronal patterning, electrophysiology and integration was collected. Original solutions were successfully applied to solve a number of issues relating to consistency of micro printing and microfluidic integration leading to successful integration of techniques and device components.
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.
An investigation into mechanisms of visceral pain in rodents
(University College Cork, 2014) Felice, Valeria D.; O'Mahony, Siobhain M.; Cryan, John F.; Irish Research Council for Science, Engineering and Technology
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.
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.
Modified cyclodextrins as novel non-viral vectors for neuronal siRNA delivery: focus on Huntington’s disease
(University College Cork, 2014) Godinho, Bruno M. D. C.; O'Driscoll, Caitríona M.; Cryan, John F.; Science Foundation Ireland; Irish Drug Delivery Network
Huntington’s Disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a mutant Huntingtin (muHTT) protein. Therefore, preventing the expression of muHTT by harnessing the specificity of the RNA interference (RNAi) pathway is a key research avenue for developing novel therapies for HD. However, the biggest caveat in the RNAi approach is the delivery of short interfering RNA (siRNAs) to neurons, which are notoriously difficult to transfect. Indeed, despite the great advances in the field of nanotechnology, there remains a great need to develop more effective and less toxic carriers for siRNA delivery to the Central Nervous System (CNS). Thus, the aim of this thesis was to investigate the utility of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as non-viral vectors for siRNA delivery for HD. Modified CDs were able to bind and complex siRNAs forming nanoparticles capable of delivering siRNAs to ST14A-HTT120Q cells and to human HD fibroblasts, and reducing the expression of the HTT gene in these in vitro models of HD. Moreover, direct administration of CD.siRNA nanoparticles into the R6/2 mouse brain resulted in significant HTT gene expression knockdown and selective alleviation of rotarod motor deficits in this mouse model of HD. In contrast to widely used transfection reagents, CD.siRNA nanoparticles only induced limited cytotoxic and neuroinflammatory responses in multiple brain-derived cell-lines, and also in vivo after single direct injections into the mouse brain. Alternatively, we have also described a PEGylation-based formulation approach to further stabilise CD.siRNA nanoparticles and progress towards a systemic delivery nanosystem. Resulting PEGylated CD.siRNA nanoparticles showed increased stability in physiological saltconditions and, to some extent, reduced protein-induced aggregation. Taken together, the work outlined in this thesis identifies modified CDs as effective, safe and versatile siRNA delivery systems that hold great potential for the treatment of CNS disorders, such as HD.
Novel strategies to enhance bumetanide concentrations in the brain in the treatment of neonatal seizures
(University College Cork, 2015) Donovan, Maria D.; Cryan, John F.; Boylan, Geraldine B.; Griffin, Brendan T.; Irish Research Council
Seizures are a prevalent neurodevelopmental disorder that affect up to 5/1000 newborns. Seizures result in detrimental developmental outcomes for many neonates, including mortality, disability and epilepsy. Current antiepileptic treatments, such as phenobarbital, display poor efficacy rates. Gamma-aminobutyric acid (GABA)-mediated signalling may promote excitatory neurotransmission in neonates with a birth injury due to intracellular accumulation of chloride. Bumetanide is a potential adjunct treatment for neonatal seizures, which reduces intracellular chloride concentrations by inhibiting the sodium-potassium-chloride cotransporter NKCC1. This accelerates the excitatory to inhibitory switch in GABA neurotransmission. In vitro bidirectional permeability assays were used to determine the apparent permeability of bumetanide and the potential for bumetanide efflux by organic anion transporter 3 (OAT3). Bumetanide was found to be a transported efflux substrate of human OAT3, thus OAT3 inhibition is a potential therapeutic augmentation strategy. OAT3 inhibition was investigated in vivo using an integrated intracerebral microdialysis model for the potential to augment bumetanide concentrations in the brain. Bumetanide was detected in brain extracellular fluid and this concentration increased after probenecid administration. An in vivo model of hypoxic-ischaemic neonatal seizures was established to quantify the pharmacodynamic effect of bumetanide in seizures. OAT3 inhibition enhanced the concentration of bumetanide in the brain in this model. Seizure burden in these animals was reduced significantly by phenobarbital and bumetanide and decreased further when an OAT3 inhibitor was administered. Physiologically-based pharmacokinetic modelling was employed to predict plasma and brain concentrations of bumetanide in a virtual neonatal population. The simulated brain tissue concentrations of bumetanide in neonates were below the half-maximal inhibitory concentration for the target transporter NKCC1. However, large interindividual variability and a paucity of physiological limited the accuracy of these simulations. In summary, augmentation strategies that focus on preventing bumetanide efflux from the brain via OAT3 may contribute to an improved outcome for neonates with seizures.
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.
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.
Novel insights into microbial regulation of transcriptional pathways in the brain
(University College Cork, 2016) Hoban, Alan E.; Cryan, John F.; Clarke, Gerard; Science Foundation Ireland; National Alliance for Research on Schizophrenia and Depression
To date, there is rapidly growing evidence that the intestinal microbiota interacts with the host at many different levels to regulate physiology. The discovery that changes in the composition of the gut microbiota correlates with alteration in brain and behaviour has largely contributed to the extension of the well characterized gut-brain axis to encompass the intestinal microbiota as a partner in gut-brain signalling, coining the term microbiota-gut-brain axis. Understanding the impact of the gut microbiota on host health and the potential mechanisms mediating these changes have largely relied on preclinical models of microbiota manipulation. Proof-of principal experiments using germ-free (GF) animals have been instrumental in establishing our current understanding of host-microbe interaction and the impact on behaviour, especially related to anxiety. This thesis investigates the underlying transcriptional changes in GF mice in key brain structures such as the amygdala and prefrontal cortex (PFC) in order to identify molecular pathways that may underlie the observed behavioural phenotype in these animals. To compliment this, we further investigated whether post-transcriptional regulatory mechanisms are recruited by the gut microbiota. Indeed, we find unique transcriptional and posttranscriptional profiles in these brain regions in GF animals and microbiota depleted animals. These findings reinforce the concept that microbes act at the molecular level to influence development and function of the amygdala and PFC, which are critical for feelings of fear and anxiety. Here we show, to our knowledge, what is the first demonstration that the microbiota is key for normal cortical myelination. Further efforts into understanding the interaction between microbes and CNS myelination may allow the development of strategies to promote remyelination in disorders of demyelination like multiple sclerosis. To further interrogate the importance of the gut microbiota in amygdala dependent behaviours we investigated whether absence of the intestinal microbiota impacted fear memory learning. We demonstrate that life without microbes results in impairments in amygdala dependent learning. Finally, this thesis demonstrates that targeted depletion of the gut microbiota in adulthood, after normal microbial assembly dramatically reshapes the behavioural and neurochemical profile and results in cognitive impairments, increased depressive-like behaviours and visceral hypersensitivity. Overall this thesis has greatly contributed to our current knowledge surrounding the importance of functional microbiota-gut-brain axis signalling and implicates the gut microbiota as a novel target for regulating cortical myelination, controlling transcriptional pathways in the amygdala and the associated expression of anxiety-like behaviours. These studies have implications for a range of neuropsychiatric disorders and lay the groundwork for therapeutic targeting of the gut microbiota.
Non-motor symptoms in the aav-α-synuclein rat model of Parkinson’s disease: exercise as a therapeutic intervention
(University College Cork, 2017) Dolan, Erin; Sullivan, Aideen M.; Nolan, Yvonne M.; Molecular Medicine Ireland
Parkinson’s disease (PD) is no longer primarily classified as a motor disorder due to the emergence of a number of non-motor symptoms (NMS) of the disease. These NMS are highly prevalent and greatly affect the quality of life of patients with PD. Thus, an animal model that replicates these symptoms is greatly needed to enhance the translational impact of preclinical research. The AAV-α-synuclein rat model is the only animal model to date that has been shown to robustly and consistently reproduce the primary neuropathological and behavioural features of PD. However, there has been little research on the ability of the model to replicate NMS of the disease. Moreover, this model is most commonly employed unilaterally, which can confound cognitive testing due to contralateral functional compensation. Thus, the aim of this thesis was to use an AAV2/6 viral vector overexpressing human wild-type α-synuclein to characterise NMS of PD, exploring behavioural phenotypes of both unilaterally- and bilaterally-administered αsynuclein. Furthermore, it set out to explore whether voluntary exercise could ameliorate motor and NMS in this PD model, including if exercise could protect against hippocampal-associated cognitive deficits by modulating adult hippocampal neurogenesis. We demonstrated that unilateral and bilateral administration of AAV-αsynuclein induced distinct patterns of nigrostriatal degeneration and associated motor dysfunction. Overexpression of AAV-α-synuclein was used to model NMS associated with PD, including deficits in hippocampalassociated tasks. This was coupled with α-synuclein-positive immunostaining in the dentate gyrus of the hippocampus, confirming the propagation of the protein throughout distinct regions of the brain. Bilateral intranigral administration of AAV-α-synuclein was found to induce motor dysfunction and a significant loss of nigral dopaminergic neurons, neither of which were rescued by voluntary running. Overexpression of α-synuclein also resulted in significant impairment on a neurogenesis-dependent pattern separation task, as well as anxiety-like behaviours on both the open field and the elevated plus maze. Voluntary running improved performance on the pattern separation task only. This was substantiated by an effect on hippocampal neurogenesis levels in the dorsal, and not ventral, dentate gyrus, suggesting that the functional effects on pattern separation were mediated by increasing neurogenesis.
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.
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.
Therapeutic potential of modulating the microbiota-gut-immune-brain axis for stress and neurodevelopmental disorders
(University College Cork, 2018) van de Wouw, Marcel; Cryan, John F.; Dinan, Timothy G.

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