Centre for Neuroscience - Theses
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ItemRelaxin-3 systems in brain: effects on feeding, anxiety, depression and addiction( 2012)The neuropeptide, relaxin-3 (RLN3), and its major endogenous receptor, RXFP3 (relaxin family peptide receptor 3), have been postulated to modulate feeding, anxiety- and depressive-like behaviour, and reward based on neuroanatomical and neurochemical data. The major aim of the research described in this thesis, therefore, was to determine the effect of centrally administered RXFP3-selective peptides on these functions in the adult rat. Intracerebroventricular (icv) administration of the RXFP3-selective agonist peptides, R3/I5 and RXFP3-A2, and native rat/mouse relaxin-3 (rmRLN3) significantly increased acute food intake in male Sprague-Dawley rats, and this effect was prevented by prior icv injection of the RXFP3-selective antagonist peptides, RXFP3-A3 and R3(B1-22)R. Icv injection of RXFP3-A2 decreased anxiety-like behaviour in male Sprague-Dawley rats in a range of behavioural tests, including the light-dark box and elevated plus maze, and produced antidepressant-like effects in the repeat forced swim test, but notably only in rats that had previously been tested in anxiety-like paradigms. Icv injection of RXFP3-selective agonists had no significant effect on general activity assessed quantitatively in automated locomotor cells. Icv administration of an RXFP3 antagonist (R3(B1-22)R) decreased alcohol but not sucrose self-administration in male inbred alcohol-preferring (iP) rats in a dose-related manner, and decreased cue-induced reinstatement (a model of relapse) for alcohol but not sucrose, suggesting a possible role for relaxin-3/RXFP3 signalling in alcohol-seeking. In addition, endogenous relaxin-3 mRNA expression in the hindbrain nucleus incertus correlated with daily alcohol and sucrose intake in the two-bottle choice paradigm, suggesting a role for endogenous relaxin-3 in modulating intake of ‘rewarding’ substances. Overall, the results from this research suggest that the endogenous relaxin-3/RXFP3 system promotes ‘motivated’ or ‘goal-seeking’ aspects of behaviour. These studies contribute to a deeper understanding of the neurobiology underlying clinical disorders such as obesity, anxiety, depression and addiction, which may in turn lead to novel diagnostics and therapeutic approaches. These important findings are therefore predicted to have a significant impact within the fields of neuropeptide biology and behavioural neuroscience, and are associated with several published or submitted scientific journal articles and reviews. In addition, the data have helped stimulate interest in this research area, with several major pharmaceutical companies expressing interest in the relaxin-3/RXFP3 system as a possible target for the development of therapeutic treatments for individual and co-morbid psychiatric and metabolic disorders.
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ItemDepression in Huntington's disease: Modulation of environment and gender( 2012)Of the plethora of symptoms that arise in HD, depression is the most diagnosed psychiatric symptom, with 30-50% of patients developing depression. This makes depression far more prevalent in the HD population than in the general populace and indeed, significantly more conspicuous compared to other neurodegenerative diseases such as Parkinson’s or Alzheimer’s disease. Due to depression’s ubiquity, impact and also the fact that it can appear decades before crippling motor symptoms, treatment and management of depression would significantly prolong the symptom-free period of patients’ lives. The overrepresentation of depression is most likely an endemic reflection of pathophysiology caused by the HD mutation. However, the aetiology of depression in the context of HD has not been well understood. The hypothalamic-pituitary-adrenal (HPA) axis is the major endocrine system responsible for stress adaptation and its dysfunction has been implicated in clinical depression. Few studies have examined the HPA-axis in HD to date. Our group has previously found, using the R6/1 mouse model of HD, a female-specific depression-like behavioural phenotype. In this thesis, examining the HPA-axis in these animals, it was found that female, but not male, R6/1 mice displayed a hyperactive HPA-axis in response to stressors. Further pharmacological challenges, gene expression analyses and in vitro studies discerned the source of the abnormality to a hypersensitive adrenal gland; a novel finding of a peripheral source for what has been largely seen as a centrally mediated pathology. Environmental factors have been found to produce significant modulation to the progression of HD. Previously, our lab has shown that environmental enrichment is able to delay the onset of various symptoms in the R6/1 mice, including depression-like behavioural phenotype in the female mice. In this thesis, environmental enrichment was also found to be able to rescue the abnormalities of the HPA-axis both in vivo and in vitro. One possible mechanism of such rescue by environmental enrichment is mediated through increased glucocorticoid receptor gene expression in the adrenal gland. Furthermore, experiments were carried out to dissect the influences of sex hormones toward explaining the sexually-dimorphic manifestation of this phenotype. Abnormality of the hypothalamic-pituitary-gonadal axis was found in both male and female R6/1 mice. Ovariectomy altered HPA-axis response in female mice, correlating with female R6/1 specific increases in estrogen receptor α gene expression in the adrenal gland. The findings in this thesis are the first to establish a peripheral origin of the HPA-axis dysfunction. It is also a first in showing that environmental enrichment can exert peripheral specific benefits. Sex-specificity of this phenotype is also a novel observation and may be due to female-specific adrenal alterations of estrogen receptor α gene expression. Clinical implications of these findings include prospective new biomarkers as well as opening up the way for potentially new targets for future treatments. Furthermore, these findings raise the consciousness regarding the importance of peripheral changes in HD and the importance of sex dimorphism in the disease progression of HD, two areas that have not been well studied.
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ItemThe role of Nedd4 family interacting protein 1 (Ndfip1) in neuronal survival following cerebral ischemia( 2010)Ischemic stroke and stroke-related illness are among the highest causes of life lost to death and disability worldwide, with substantial social and economic costs. Despite significant progress in understanding the pathophysiology of ischemic stroke, and wide-scale clinical testing of promising experimental agents, very few patients have access to effective treatment. The need for more efficacious therapeutic options and the failure to translate neuroprotection from experimental studies to human disease has led to a re-evaluation of therapeutic research at all levels. As a way forward, there has been a fundamental shift towards studying the brains' own intrinsic survival response to ischemic stress. Evidence suggests that the brain can evoke gene expression changes and biochemical modifications in response to ischemia that provide endogenous neuroprotection. Accordingly, the identification of these protective mechanisms may provide better therapeutic targets for ischemic stroke. Our laboratory previously identified Ndfip1 (Nedd4 family interacting protein 1) as an endogenous protein upregulated in response to acute traumatic brain injury and associated with neuronal survival. In the brain, Ndfip1 is neuron-specific and acts as a regulator/adaptor for the Nedd4 family of ubiquitin E3 ligases that catalyse the post-translational ubiquitination of proteins. While Ndfip1 and its homologs are known to regulate a number of physiological processes by promoting the ubiquitination of Nedd4 target substrates, it is also becoming apparent that Ndfip1 is a highly conserved stress response protein. In partnership with Nedd4 E3s, Ndfip1 and its yeast homolog confer protection against acute cellular injury induced by different pathological conditions, including brain trauma, heavy metal toxicity, growth factor starvation, hyperthermic stress and canavanine toxicity. As the pathological basis of these injury paradigms is related to ischemia pathogenesis, it was hypothesised that Ndfip1 is a potential component of the intrinsic survival response to ischemic stroke. Furthermore, as the protective effect of Ndfip1 in other injury paradigms is linked to its capacity to promote Nedd4 family-mediated ubiquitination, it was also hypothesised this mechanism could underlie potential neuroprotection by Ndfip1 in ischemia, implicating the essential involvement of Nedd4 E3s. To test these hypotheses, this thesis examined the role of Ndfip1 in neuronal survival following cerebral ischemia and studied the involvement of Ndfip1-mediated ubiquitination and Nedd4 family proteins known to interact with Ndfip1. Using endothelin-1-induced middle cerebral artery occlusion (ET-1-induced MCAo) in rats and hypoxia-ischemia (HI) in mice, these two distinct models of cerebral ischemia revealed endogenous Ndfip1 was consistently upregulated in peri-infarct cortical neurons during infarct development. In the ET-1 model, the topographic distribution of Ndfip1 upregulation in cortical peri-infarct regions followed a predictive pattern that was coincident with infarct progression, and the extent of Ndfip1l upregulation was dependent on stroke severity and post-ischemia duration. In the HI model, an analogous response was observed in the cortex, while Ndfip1l upregulation was also detected in hippocampal and thalamic neurons known to be susceptible to HI-induced injury. Overall, there was a strong positive correlation observed between the number of TUNEL-positive cells and the number of Ndfip1-upregulated neurons, and taken together with the above findings, suggested Ndfip1 upregulation was an injury-related response modulated by the degree and severity of ischemia. Nonetheless, Ndfip1-upregulated neurons in peri-infarct cortex did not colocalise with the majority of TUNEL-positives cells following ET-1-induced stroke, suggesting Ndfip1 upregulation is strongly correlated with neuron survival and represents an endogenous neuroprotective response. Consistent with this notion, overexpression of Ndfip1 conferred protection against oxygen and glucose deprivation (OGD) in vitro in PC12 and SH-SY5Y cell lines. Furthermore, loss of Ndfip1 expression in Ndfip1+/- hypermorph mice exacerbated their susceptibility to cortical and hippocampal infarction following hypoxia-ischemia in vivo. Together, these in vitro and in vivo studies suggest that Ndfip1 promotes neuron survival in response to cerebral ischemia. Characterisation of several Nedd4 family members in the ET-1 model revealed that Nedd4-2 was specifically co-upregulated with Ndfip1 in ischemic stroke, and immunoprecipitation assays confirmed that Ndfip1 and Nedd4-2 could interact in physiological and ischemic conditions. Conversely, other known Ndfip1-binding partners from the Nedd4 family were not co-regulated with Ndfip1 following ischemia. While Nedd4 was not differentially expressed following ET-1-induced MCAo, Itch underwent upregulation/translocation in response to ischemia, but this response did not colocalise with Ndfip1 upregulation, suggesting the relationship between Ndfip1 and Nedd4-2 was specific in ischemic stroke. In the presence of endogenous Nedd4-2, induction of Ndfip1 overexpression that conferred survival against OGD was also associated with increased ubiquitination in vitro, suggesting Ndfip1-mediated ubiquitination via Nedd4-2 is linked to the neuroprotective mechanism of Ndfip1 in cerebral ischemia. Collectively, the results presented in this thesis suggest that Ndfip1 is an endogenous stress response protein that promotes neuron survival in cerebral ischemia through Nedd4-2-mediated ubiquitination. These findings provide new insight into the regulation and function of Ndfip1 and Nedd4-2 in pathological conditions, and highlight the importance of ubiquitination in the endogenous survival response to cerebral ischemia. Importantly, these findings suggest Ndfip1/Nedd4-2-mediated ubiquitination may provide a potential target for the treatment of clinical ischemic stroke.
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ItemThe control of Nedd4 family interacting protein 1 (Ndfip1) expression and its binding partners( 2012)The Nedd4 family interacting protein 1 (Ndfip1) is a neuroprotective protein, highly up-regulated in the neuron following brain injury. Many fundamental questions regarding the functions and regulations of Ndfip1 remained unsolved. Therefore, this study aimed to investigate Ndfip1 from three different aspects: 1) novel drugs or compounds which up-regulate the Ndfip1 levels, 2) novel binding partners of Ndfip1 and 3) specific functions and downstream processes of following the interaction between Ndfip1 and PTEN. A high-throughput screening method was developed in this study in order to search the novel drugs or compounds which up-regulate Ndfip1 level. Funneling from the large collection of bioactive compounds (4400 compounds), this process had short-listed 23 compounds which potentially up-regulate the levels of Ndfip1. Six compounds from the short-listed potential candidates were validated and successfully up-regulate Ndfip1 levels in various cell lines. The search of novel Ndfip1 binding partners involved with a two-step purification technique, known as tandem affinity purification. By using Ndfip1 as a bait protein, a large network of protein candidates binding with Ndfip1 was identified. Among these proteins candidates, the FANCD2 (Fanconi anemia group D2 protein) was validated using Duolink technique, and its interaction with Ndfip1 was confirmed. FANCD2 is a pivotal protein involved in DNA damage signaling and repair. The association between Ndfip1 and FANCD2 suggest that Ndfip1 might play a role in regulating the DNA damage signaling and repair. The most prominent impact derived from this study is the discovery of Ndfip1 and PTEN interaction. PTEN is a tumor suppressor protein which involved in the tumor formation if PTEN is mis-regulated. In recent years, more reports associated PTEN with the role of neuroprotection. This interaction between Ndfip1 and PTEN enhanced PTEN ubiquitination. Ndfip1 mediated PTEN ubiquitination resulting in PTEN nuclear translocation, but not degradation. Furthermore, this Ndfip1-PTEN interaction is dominantly regulated by PTEN phosphorylation. The contributions of this study include gaining better understanding regarding the control of Ndfip1 levels, as well as the network of proteins which bind to Ndfip1. This study also provided a mechanistic understanding regarding the neuroprotective function of Ndfip1. Here, I would like to propose that the increased level of Ndfip1 during neuronal injury, leads to PTEN nuclear translocation, and promote cell survival through the regulation of p-Akt levels in stressed neurons. Therefore, it will be in great interest to follow-up the leads from this study, in order to understand in greater detail the specific neuroprotective mechanism of Ndfip1.
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ItemIdentification of cellular responses to autoimmune injury induced in neurons( 2012)Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that is pathologically characterized by focal inflammatory lesions featuring demyelination, gliosis and axonal injury. Immune-mediated axonal damage and loss are accepted as major determinants of irreversible neurological disability in MS patients. However, it remains unclear how inflammatory cells damage axons in acute MS lesions, and whether axons or neurons respond to this injury to limit its extent. Here we used a common mouse model of MS pathology, murine MOG35-55 experimental autoimmune encephalitis (EAE) to characterize potential endogenous responses to autoimmune injury in neurons. The MOG-EAE model features marked inflammatory axonal injury in the spinal cord encompassing axons that originate from corticospinal motoneurons, located in the motor and sensory cortex of the brain. To reveal specific neuronal responses to axonal damage in the spinal cord, we compared gene expression in cortical regions of EAE and healthy control mice by microarray. Although inflammatory infiltrates were absent in the cortex, we detected gene expression alterations in EAE mice. Unexpectedly, many of these genes encoded for proteins that were functionally associated with the extracellular matrix (ECM). Expression of the ECM adaptor molecule, matrilin-2 (Matn2), correlated with EAE disease severity and was specifically increased in cortical areas projecting to the spinal cord. In addition, Matn2 protein was expressed by neurons in these regions. In the spinal cord of EAE mice, Matn2 was detected within acutely damaged axons in developing lesions, and with disease progression, was predominantly found extracellularly in and around the lesions. These observations suggest that Matn2 is regulated following neuronal damage and is subsequently released into the ECM. In addition, Matn2 markedly accumulated in the perivascular space of inflamed blood vessels. Importantly, in tissue samples of MS patients, Matn2 was also detected in neurons and Matn2 depositions were found in acute and chronic lesional parenchyma as well as in perivascular areas, suggesting a potential relevance of Matn2 to MS pathology. Functional analyses using Matn2 knockout (ko) mice in EAE showed an ameliorated disease severity at the acute disease stage. This coincided with smaller and fewer lesions, reduced axonal injury and decreased expression of genes encoding markers of microglial/macrophage activation (CD45 and CD11b) and pro-inflammatory molecules predominantly expressed by activated macrophages (Il-1b, Tnfa, Il-6, Cox-2 and iNos). Although, the number of immune cells in spinal lesions was unchanged in wt and ko EAE mice with equal (matched) EAE grades, expression of these genes was still reduced in ko mice. This led us to hypothesize that Matn2 can modulate microglial/macrophage activation and induce pro-inflammatory gene expression. In vitro studies using a neuron/macrophage co-culture model showed that Matn2 gene expression increased in cortical neurons following acute axonal injury by activated macrophages. Importantly, addition of recombinant Matn2 to neuron/macrophage co-cultures resulted in macrophage activation and induced pro-inflammatory cytokine expression as well as in acute axonal injury. These results support the described in vivo findings and give insight into a potential mechanism of Matn2-mediated axonal damage. Collectively, the present study introduces Matn2 as a novel mediator of neuroinflammatory disease and the innate immune response, in the EAE model. We show that Matn2 is an endogenous neuronal protein that is induced and potentially secreted/released following acute axonal injury. We found that Matn2 can modulate macrophage activation to promote pro-inflammatory activity, and consequently, axonal injury in acute inflammatory lesions. Strikingly, deletion of Matn2 significantly ameliorated disease severity and reduced axonal damage. Therefore, targeting Matn2 in the acute lesion environment presents a potential therapeutic strategy in MS pathology.
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ItemMolecular mechanisms of Ndfip1 during brain development and consequences for neuronal survival( 2012)In this thesis, I describe experiments to study the function of Ndfip1 (Nedd4 family-interacting protein 1), an adaptor for Nedd4 family of ubiquitin ligases, during development of the mouse neocortex and its response following traumatic brain injury (TBI). Ndfip1 functions by binding and recruiting proteins for ubiquitination by Nedd4-family ubiquitin ligases, comprising nine members. Ubiquitinated proteins can be either degraded in the proteasome apparatus or become signaling proteins. Either way, it has a profound impact on the subsequent behaviour of neurons during development or disease. In the present study, I first investigated the spatial and temporal expression pattern of Ndfip1 during cortical development. In the early stages (embryonic day 11, E11), Ndfip1 is highly expressed in proliferative cells of the germinal zone where neurons are born. As development progresses (E15 onwards), Ndfip1 expression shifts to the mature neurons in the cortical plate, with concomitant reduction in the ventricular zone. This dynamic shift from proliferative to non-proliferative regions of the cortex suggests a dual role for Ndfip1 in proliferating neurons and mature neurons. To explore this, I investigated the relationship between Ndfip1 and Sprouty2 (Spry2), an inhibitor of cell division and cell survival via the MAP-kinase pathway. I provide evidence to demonstrate that Ndfip1 binds to Spry2, in both endogenous and over-expression systems. In the developing cortex, Ndfip1 and Spry2 expression are similar and coincidental. In a neuronal cell line (SY5Y), artificial over-expression of Ndfip1 results in reduction of Spry2, suggesting that Ndfip1 can down-regulate Spry2 levels most likely by Nedd4 ubiquitination. Consistent with this notion, the levels of Spry2 are upregulated following Ndfip1 loss in Ndfip1-/- fibroblasts. This upregulation of Spry2 is associated with attenuated epidermal growth factor-elicited ERK1/2 signaling. Therefore, association of Ndfip1 with Spry2 might be important for the regulation of of Spry2 and the MAP-kinase signaling pathway during cortical development. I have also investigated the potential role of Ndfip1 as a neuroprotective agent following brain injury by using a mouse model of closed head injury. Ndfip1 was upregulated in surviving neurons close to the trauma lesion at 6 h and 24 h post-TBI. Given that Ndfip1 can bind and mediate ubiquitination of the tumor suppressor PTEN, I investigated the relationship between the two. I demonstrate, for the first time, that Pten is translocated to the neuronal nucleus following brain injury. This event is coincident with Ndfip1 upregulation and survival of neurons situated close to the sites of lesion. I performed biochemical assays that revealed that Pten levels remained stable after TBI, suggesting that nuclear Pten in cortical neurons results from relocation of existing cytoplasmic Pten rather than Pten upregulation. In vivo, I also show that Ndfip1 upregulation and Pten nuclear trafficking are events associated with neuronal survival after TBI, as mice lacking Ndfip1 sustained larger brain lesions compared to wild-type controls. In addition Ndfip1 upregulation is correlated with increased Akt phosphorylation in the trauma hemisphere, suggesting that neuron survival is associated with higher p-Akt levels. Finally, I demonstrate that TBI induces increase binding of Ndfip1 to Nedd4-2, but not Nedd4-1 indicating that Nedd4-2 is the E3 ligase for Ndfip1 in the brain. I conclude that in brain injury, Ndfip1 together with Nedd4-2 is neuroprotective in surviving neurons by trafficking Pten into the nucleus, rather than by degrading cytoplasmic Pten. These experiments firmly establish that, through Pten, Ndfip1 is a key regulator of PI3-kinase signaling for cell survival following brain injury. In summary, the experiments in my thesis provide novel evidence that Ndfip1 is an important player during development of the cortex in the embryo, and protection of the cortex in the adult. In either scenario, Ndfip1 functions by regulating the MAP-kinase and PI3-kinase pathways, which are known to control a multitude of cellular functions including cell growth and cell survival. I provide strong evidence to suggest that Ndfip1 regulates the negative regulators (PTEN and Spry2 respectively) of PI3-kinase and MAP-kinase signaling pathways. My work offers the foundation for future strategies to manipulate Ndfip1 for improving neuron survival.
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ItemModulation of bone morphogenic protein signalling alters demyelination and myelin repair in the corpus callosum of adult mice( 2012)Multiple Sclerosis (MS) is the most common progressive neurodegenerative disease of young adults. A key pathological event in MS is oligodendrocyte apoptosis, which leads to axons losing their myelin sheaths throughout the central nervous system (CNS). Therefore, enhancement of oligodendrocyte regeneration by endogenous progenitor cells is a promising strategy for remyelination. Bone morphogenic proteins (BMPs) are a family of growth factors which have been shown to inhibit oligodendrocyte production in the healthy CNS. This thesis describes the role for BMP signalling in demyelination and remyelination in the adult brain. Firstly, the cellular changes in the subventricular zone (SVZ) are examined during cuprizone-induced demyelination and in response to BMP signalling modulation. BMP signalling is active in the SVZ during demyelination, and infusion of the BMP4 antagonist, Noggin, decreased the number of astrocytes and increased the number of oligodendroglial cells. Work from this thesis has also identified that BMP signalling is active in the demyelinated corpus callosum. BMP4 infusion increased oligodendrocyte progenitor cell (OPC) numbers in the demyelinated corpus callosum, while infusion of Noggin increased numbers of mature oligodendrocytes and remyelinated axons following recovery from cuprizone challenge. Furthermore, sequential infusions of BMP4 and Noggin or BMP4 and insulin-like growth factor-1 (IGF-1) were performed during demyelination to determine whether myelin repair could be further enhanced by delivery of BMP4, to increase the pool of OPCs, followed by Noggin or IGF-1 to increase either differentiation or survival of the OPCs. Sequential delivery of BMP4 and Noggin or IGF-1 does not further enhance myelin repair above what occurs with Noggin infusion alone. The results in this thesis indicate an important role for BMP signalling in modulating oligodendrogliogenesis and remyelination. The research described here has identified BMPs as potential therapeutic targets to enhance myelin repair in MS.
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ItemModels of brain targeted D1 dopamine receptor positive cell ablation( 2012)Dopamine receptor D1a (Drd1a) is one of the most abundant dopamine receptor subtypes in the central nervous system and is implicated in many neurological processes including neuronal growth, development and behavioral responses. Previously, we reported that mice with targeted ablation of Drd1a receptor-positive cells (CamKIIαtox MUT) exhibited behavioral abnormalities similar to those observed in models of Huntington's Disease (HD). In the present study, we focused on identifying the pathological locus for HD-related behavioral deficits by characterizing neurochemical changes within CamKIIαtox MUT mice further, as well as generating a new transgenic line with restricted Drd1a ablation only to the cortex. This novel transgenic line utilized the same Cre-Lox system employed in our previous study but replacing the CamKIIαCre delivery line with an Emx-1Cre line to generate mutant (Emx-1tox MUT) mice that lacked Drd1a-expressing cortical pyramidal cells. Adult Emx-1tox MUT mice with intact Drd1a expression and striatal markers were hyperactive, displayed forelimb dominant clasping, poor rotarod performance, heightened anxiety-like behaviors and impaired memory function. As hypothesized, we found Emx-1tox MUT mice to demonstrate reduced cortical thickness compared to controls in both motor and somatosensory domains, particularly within the deeper cortical layers. This correlated strongly with the loss of DARPP-32-expressing cells in the same region. This study demonstrates a primary role of cortical Drd1a expressing cells in motor control and cognition.
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ItemCharacterisation of insulin-regulated aminopeptidase in the brain: novel roles in memory and Alzheimer's disease( 2012)Insulin-regulated aminopeptidase (IRAP) is a type II membrane bound metalloprotease, which in the brain is expressed in neurons important for cognitive function. Inhibitors of IRAP elicit dramatic effects upon central administration in rodents, including improved learning and memory performance, and attenuation of drug and lesion-induced memory deficits. Non-peptide IRAP inhibitors with improved pharmacokinetic profiles have been developed to improve specificity and efficacy. However, the role of IRAP in the brain is yet to be firmly established, and the mechanistic link between IRAP inhibition and memory enhancement is under much debate. The aim of this thesis was to identify novel roles of IRAP in the normal and pathological brain, which would support further development of IRAP inhibitors in the treatment of memory deficits. I first characterised the behavioural phenotype of the forebrain neuron-specific postnatal IRAP knockout mouse (fb KO) to determine the role of IRAP in the adult brain. Despite apparently normal gross brain anatomy, young adult male fb KO mice were unable to perform two hippocampus dependent reference memory tasks, whilst wild type littermates scored well above chance in both paradigms. As the spatial reference memory deficits were absent if fb KO animals were maintained in social groups, cognitive function in the IRAP fb KO mice appears to be more sensitive to home cage conditions. This exaggeration of environmental effects on memory could be due to altered hippocampal neurogenesis. This correlates with previous findings in the global IRAP-/- mouse, and strengthens the case for an important role for early expression of IRAP in memory function in the adult. Whilst IRAP is exclusively a neuronal protein in the normal brain, preliminary evidence was available for the expression of IRAP in activated astrocytes after traumatic brain and spinal injury in laboratory animals, and in the brains of Alzheimer’s disease (AD) patients. I confirmed in two mouse models of AD that IRAP was extensively expressed in activated astrocytes associated with amyloid pathology, throughout the cortex and hippocampus. IRAP was also prominent in activated astrocytes of the aged mouse brain and in activated astrocytes in vitro. Current treatments for Alzheimer’s disease, the leading cause of dementia in Australia, are restricted in scope and offer modest symptomatic relief. IRAP inhibitors are potent cognitive enhancers, yet prior to this study they had not been tested for efficacy in a model of AD. I studied the effects of IRAP inhibition in the 5xFAD early onset and the Apd9 late onset mouse models of AD. I showed promising evidence that chronic IRAP inhibition could attenuate spatial deficits in the 5xFAD model, and object recognition deficits in the Apd9 model, both paradigms that reflect the cognitive domains impaired in early AD. Surprisingly, treated 5xFAD mice also demonstrated preliminary evidence of changes to underlying neuropathology, including increased microglial activity and reduced plaque load in the dorsal subiculum. Thus, IRAP inhibition may offer improvements over current cognitive enhancers due to the concomitant modulation of pathology. Collectively these results suggest the potential involvement of IRAP in two key processes in the brain: neurogenesis and astrogliosis, both of which are important in different stages of brain development. Manipulation of IRAP activity using specific IRAP inhibitors has revealed the exciting potential for these compounds in the development of disease modifying treatments for Alzheimer’s disease, a major area of drug development.
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ItemEarly gene expression following peripheral nerve injury( 2011)The mammalian nervous system regulates the response to both internal and external stimuli, thus any injury can have profound effects leading to considerable disability. Although advancements in surgical technique have facilitated significant improvements in the treatment of peripheral nerve injuries, there have been few recent innovations leading to further addditional improvement in functional outcome. As such, attention has now turned to understanding the inherent cellular and molecular mechanisms that underlie the regeneration response. This thesis will attempt to improve the understanding of these processes that are activated within the first 24 hours following peripheral nerve injury. It is proposed that discernable patterns can be elucidated to enable a deeper understanding of the biological processes that are modulated following traumatic neuronal injury, and appropriate therapeutic targets could be developed to improve the functional outcome. The rodent sciatic model was used to facilitate whole genome microarray analysis of gene expression profile of the dorsal root ganglion (DRG) following axonal injury. During ultra-early temporal analysis (less than 24 hours after injury), differential gene expression occurred within four hours and peaked at 24 hours after axotomy, with 2,022 probe-sets determined to be significant. One biological pathway modulate was the mitogen-activated protein kinase (MAPK) cascade that is fundamental for neuronal survival and regeneration. As the pattern of change in mRNA cannot be directly correlated with the biological function of related proteins, phosphorylation of two effector proteins of the MAPK cascade at 4 and 24 hours, c-Jun (76.8+/-2.7%; p<0.05 and 80.6+/-4.1%; p<0.01) and ATF2 (72.7+/-1.2%, p<0.01 and 82.2+/-1.4%, p<0.001) were studied and found to be modulated in a similar fashion iv within the neuronal and satellite glial cell (SGC) population respectfully. In addition, there was evidence of SGC proliferation within the first 24 hours after axotomy (0.2+/-0.01, p<0.05 and 0.2+/-0.01, p<0.005). At 24 hours post-injury, differential gene expression was found to be proportional to the severity of axonal injury (2,060 significant probe-sets). Similarly, following crush injury and axotomy, the neuronal phosphorylation of c-Jun (77.9+/-3.2%; p<0.001 and 84.6+/-4.3%; p<0.001), SGC phosphorylation of ATF2 (76.6+/-8.6%, p<0.001 and 78.6+/-6.2%, p<0.001) and SGC proliferation (0.13+/-0.01, p<0.01 and 0.14+/-0.01, p<0.001) was discovered to be also proportional to the severity of axonal injury. Following crush injury and axotomy, the glucocorticoid dexamethasone was administered and found to induce the neuronal phosphorylation of c-Jun while inhibiting the SGC phosphorylation of ATF2 and proliferation at 24 hours post-injury. What remains to be clarified is the functional repercussions of the effect of dexamethasone on these proteins and SGCs proliferation. It is proposed that the understanding of the biological processes that occur during the neuronal response to axonal injury gained within this thesis, provides the opportunity to potentially target biological processes that might improve functional outcomes following peripheral nerve injury. This project provides the platform for future studies to test means of neuro-protection following axonal injury.