Florey Department of Neuroscience and Mental Health - Theses
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ItemStem cells for the treatment of neurodegenerative disorders( 2016)Neurological disorders present a special challenge to medical science, because of their increasing prevalence, and the inability of the brain and spinal cord to repair itself. While the prevention of these conditions is preferable, the reality is that there will most likely always be a heavy dependence on therapies that treat established disease. Cell therapy holds significant promise for the treatment of these disorders, although substantial challenges remain before they can be progressed into mainstream therapies. This thesis explores some of the factors that affect the successful integration of stem cell-derived neural cells into the brain, in a series of experiments presented across four research chapters. Notably, the research has demonstrated that therapeutic neural cells derived from human iPS cells require a period of 12 months after implantation to mature, in a similar manner as what is observed in normal human development. This has implications for preclinical investigations utilising human cells to repair the damaged brain, which may require studies to run for periods of up to 1 year. These studies rely heavily on the transplantation of human therapeutic cells into discordant species, such as rodents and non-human primates. Accordingly, we conducted a systematic evaluation of the ability range of immune-modulating approaches to sustain human xenograft survival in the rat. While robust survival of hESC-derived neural cells was observed in athymic animals, survival of the same cells in immunocompetent adult or neonatal-grafted animals did not exceed 14 weeks. Immunosuppression by pharmaceutical agents resulted in cell-survival beyond 20 weeks, which was associated with a reduction in blood and brain T-cell quantities. These results demonstrate the utility of the athymic rat in xenografting studies, and provide practical information for the design of preclinical studies. When we attempted to utilise pharmacological immunosuppression to bolster grafted-cell survival in the SOD1G93A rat model of motor neuron disease, we observed a significantly detrimental impact associated with this agent on the motor performance of these animals. This highlighted the problem of immunosuppression in models of neurodegeneration, where the disease pathology is influenced by immune-effects. This presents a particular challenge to cell therapies aimed at treating these conditions, and we therefore evaluated the impact of treatment with three commonly used immunosuppressants, cyclosporin A, FK506 and rapamycin, on core disease characteristics in the SOD1G93A rat. Rotarod performance was disrupted by treatment with rapamycin and cyclosporin A, but not by treatment with FK506. The observed impairment occurred despite rapamycin reducing local expansion and recruitment of microglia, and cyclosporin A reducing levels of misfolded SOD1 protein within the spinal cord. In contrast, FK506 appeared to increase astrocyte activation, whilst not impairing behavioural outcomes. This study highlights that immunosuppression to support xenograft survival may directly affect important disease traits in models of neurodegeneration. Thus, the use of immunosuppression in such paradigms should be carefully considered within study design. While the long-term survival of implanted cells is a critical feature of successful stem cell-based therapies for neurodegenerative disorders, understanding the factors that guide the growth and integration of implanted neural cells is equally important. We performed a study that compared the fibre pathways of orthotopically and heterotopically transplanted fetal tissue-, as well as mouse embryonic stem cell- derived neural progenitors, with endogenous fibres of the intact adult murine cortex. Fetal tissue transplanted into the visual and motor cortices projected fibres across the entire dorsoventral axis of the adult brain. Quantification of the innervation of these fibres in specific targets of the cortex did not reveal an overwhelming tendency for grafted cells to target nuclei relevant to their intrinsic identity. While mouse embryonic stem cell-derived neural progenitors survived and expressed markers of mature cortical cells types in vivo, these cells did not demonstrate a high degree of axonal outgrowth. When quantified, there was no substantial difference in the innervation of specific cortical areas when mouse embryonic stem cell-derived neural progenitors were placed in either the motor or visual cortices. This suggests that the innervation patterns of implanted cells cannot be used to assign a particular areal identity to donor cells, as the passive outgrowth of fibres along host white matter tracts cannot be excluded as a possibility. Instead, immunohistochemical analysis of the expression of markers for specific areal identity may assist future studies in this purpose. Taken together, this work enhances our understanding of how certain factors must be managed to ensure the integration of donor cells in preclinical investigations, so that one day these studies can be progressed into therapies that support full patient rehabilitation.
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Item‘Somnivore’ a user-friendly platform for automated scoring and analysis of polysomnography data( 2016)The low-throughput nature of manual scoring of polysomnography (sleep) data, both in terms of speed and consistency, is a major factor preventing sleep research from reaching its full efficiency and potential. Automated approaches developed previously have generally failed to provide sufficient accuracy or 'usability' for sleep scientists lacking specialist-engineering expertise. Moreover, all earlier approaches have only been validated using baseline data, suggesting a failure to embed in the algorithm the robustness to remain effective when used to analyse the effect on sleep of treatment or disease. Finally, no single approach has been validated for mouse, rat and human data. Therefore, the aim of my research was to develop a user-friendly platform for real-time automated scoring and analysis of polysomnography data. The program is known as ‘Somnivore’ (from Latin somnus, ‘sleep’, and vorare, ‘to devour’), and was developed using state of the art supervised machine learning technology, with support vector machine (SVM) at its core, and coded as a graphical user interface (GUI)-based solution in the Matlab™ ambient. Somnivore learns, in parallel, by surveying features from a variety of different inputs (including EEG, EMG, EOG and ECG) and outputs data into the various sleep stages (wake, NREM, N1, N2, N3, REM). The classifier is trained for each subject via a brief session of manual scoring. Design and development strategies were built around both theoretical and heuristic approaches. This led to a multi-layered system capable of learning from extremely limited training sets, using large input space dimensionalities from a rich variety of polysomnography inputs, and with rapid computational times. Validation was pursued to approach the numerous contentious dynamics that have led to the demise of previous solutions. Somnivore generalisation was evaluated at the level of canonical classifier evaluation metrics such as F-measure, as well as experimental end-measures more germane to traditional biological sleep research. Somnivore, generated superior generalisation, with high power, on both murine (n = 54) and human (n = 52) recordings. These included multiple rat strains (Sprague-Dawley, Wistar) and mouse phenotypes (wild type, orexin neuron-ablated transgenic), various pharmacological interventions (placebo, alcohol, muscimol, caffeine, zolpidem, almorexant), and in humans, both genders, younger and older subjects, and subjects with mild cognitive impairment (MCI). Somnivore’s generalisation was also evaluated in conditions of signal challenged data, and provided excellent performance in all conditions using only one EEG channel for learning. Remarkable results were also reported for learning undertaken using only one EMG channel or two EOG channels. Furthermore, validation studies highlighted that a substantial part of the disagreement between manual and automated hypnograms was located within transition epochs. As Somnivore has several features geared towards the management of transition epochs, further control over generalisation is also possible. Comprehensive inter-scorer agreement analysis was conducted on human data, showcasing how inter-scorer agreement between manual hypnograms and their automated counterparts provided by Somnivore is comparable to the gold-standard of the inter-scorer agreement between two experts trained in the same laboratory. Results also highlighted critical problems within the scoring of stage N1. However, inter-scorer agreement validation studies also confirmed what has already been reported in the literature, that N1 is a volatile stage that systematically produces inadequate agreement even between trained experts, both within or outside the same laboratory. Accordingly, Somnivore performed as well on N1 as reported in the literature for manually scored data. Due to the high-throughput nature of Somnivore’s analyses of experimental end-measures, several novel, cautionary findings were extracted from the recordings provided by external laboratories for this research evaluations. Additionally, as Somnivore is also capable of scoring real-time during polysomnography recordings, it will facilitate the development of more advanced protocols such as biofeedback sleep-deprivation protocols and integrated optogenetics. In conclusion, Somnivore, has been comprehensively validated as an accurate, reliable, high-throughput solution for scoring and analysis of polysomnography data, in a range of experimental situations including studies of normal physiology and tests related to drug discovery for the improved treatment of sleep disorders and psychiatric diseases.
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ItemPrefrontal dopaminergic mechanisms of adolescent cue extinction learning( 2016)Addiction and anxiety disorders represent the most prevalent mental illnesses in young people worldwide. Unfortunately, adolescents attain poorer outcomes following extinction-based treatment for these disorders compared to adults. Cue extinction learning involves dopamine signaling via the dopamine 1 receptor (D1R) and dopamine 2 receptor (D2R) in the medial prefrontal cortex. In particular, the infralimbic cortex, a subregion of the medial prefrontal cortex, has been implicated in extinction learning in both adolescent and adult rodents. The prefrontal dopamine system changes dramatically during adolescence. However, the role of prefrontal dopamine in adolescent cue extinction learning is poorly understood. Therefore, this thesis aimed to elucidate the role of prefrontal dopamine in adolescent cue extinction, using cocaine self-administration and fear conditioning in rats. My first study examined cocaine self-administration and cocaine-associated cue extinction in adolescent versus adult rats. Adolescents displayed a deficit in cocaine-cue extinction learning compared to adults (postnatal day [P]53 and P88 on cue extinction day, respectively). A single infusion of the full D2R agonist quinpirole into the infralimbic cortex prior to extinction enhanced adolescent cue extinction to reduce relapse-like behavior the next day. This effect was recapitulated by a systemic injection of the partial D2R agonist aripiprazole, an FDA-approved drug for the treatment of psychosis with strong translational potential. My second study examined fear conditioning and extinction in adolescent and adult rats. I first aimed to optimize behavior in late adolescent (P53) and adult (P88) rats during the dark phase of their 12-hour light-dark cycle, to remain consistent with conditions of the previous chapter. However, this produced unreliable behavioral results. In contrast, adolescent rats (P35) consistently display a deficit in long-term fear extinction compared to adults (P88) during the light phase. Infusion of the D1R agonist SKF-81297 into the infralimbic cortex prior to fear extinction had no effect for either age group. However, infusion of quinpirole into the infralimbic cortex significantly enhanced long-term fear extinction in adolescents, whereas it delayed within-session extinction in adults. Interestingly, an acute systemic injection of aripiprazole improved long-term fear extinction in adults. My final experiments measured prefrontal gene expression for D1R, D2R, and D1R relative to D2R (D1R/D2R ratio) in naïve rats across adolescent development, or following cocaine-cue, or fear extinction. There were no significant differences in prefrontal dopamine receptor gene expression across naïve rats age P35, P53, and P88. Following cocaine-cue extinction, prefrontal D1R gene expression was upregulated in adults but not adolescents. By comparison, following fear conditioning, adolescents showed higher D1R and D1R/D2R ratio gene expression compared to adults. D1R/D2R ratio was modulated in opposite directions following fear extinction learning during adolescence versus adulthood. These findings show that adolescents are impaired in extinction of emotionally salient cues across both appetitive (drug) and aversive (fear) learning domains. Functional and molecular data provide novel evidence for divergent involvement of prefrontal dopamine in cue extinction learning across adolescent development. Results not only extend understandings of extinction learning in general, but represent an exciting step towards finding new therapeutic targets to facilitate exposure-based therapy in the clinic.
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ItemUnderstanding neural development and the establishment of region-specific protocols for the differentiation of naive pluripotent stem cells( 2016)Pluripotent stem cells (PSCs) have the potential to differentiate into the three germ lineages including ectoderm, and more specifically neuroectoderm, in an effort to generate different neuronal populations. These resultant neural progenitors and neurons can be utilized for various purposes, such as modeling development and/or disease in vitro, neural transplantation, as well as drug development and testing. Studies to date involving the neural differentiation of mouse PSCs have been hampered by the lack of highly defined protocols to generate specific neural populations. Hence, improved protocols to derive specific neural populations from PSCs are highly required. Chapters 3 and 4 of this PhD thesis focus on developing novel differentiation procedures to generate region-specific neurons (dorsal forebrain, ventral forebrain, ventral midbrain, hindbrain and medial ganglionic eminence) from naïve ground state PSCs. To begin, we demonstrate that the use of naïve mouse embryonic stem cells (mESCs), reflective of blastocyst pre-implantation pluripotent stem cells, improves neural differentiation in comparison to primed, postimplantation- like, mESCs. To direct the fate of neural progenitors, we have utilized a number of morphogens (such as SHH for ventralization and Wnts for caudalization) that are involved in mammalian neural development. Furthermore, the spatial and temporal expressions of the progenitors and mature neurons, derived using our novel differentiation systems, have confirmed their region-specific identity. Whilst these improved protocols enable the generation of regionally specified neural progenitors and neurons, more restricted balances between extrinsic morphogen gradients (such as those manipulated in chapters 3 and 4) together with specific intrinsic gene profiling results in the more specialized populations of neurons within each of these regions, such as the dopaminergic (DA) neurons within the ventral midbrain (VM). Despite decades of research into understanding the development of VM DA neurons, numerous key events involved in their specification and connectivity remain unknown. A recent microarray study within laboratory generated a gene list of novel genes involved in ventral midbrain. Included in this list, and examined in chapter 5 of this thesis is the role of the cell adhesion protein, Close homology to L1 (CHL1), in the birth and connectivity of midbrain dopamine neurons. Using VM primary cultures and Chl1 deficient mice, the results from this chapter show the diverse roles of both soluble and membrane-bound CHL1 in migration of DA progenitors, their differentiation and finally their connectivity. We identify that in these contexts, CHL1 acts through homophilic (CHL1-CHL1) interactions between the DA neuron and the local environment. These findings provide the first reports of a cell adhesion protein in the development of VM DA neurons. The results from these experiments could (i) improve the derivation of specific neuronal populations, hence, improving their use in drug testing, transplantation and disease modeling. For example, MGE-specific progenitors and mature neurons, derived using our protocol, could be used to study epilepsy or could be used for drug screening and development. In addition, transgenic lines could be differentiated using our protocols to region-specific neurons to understand their development in vitro. The results from these experiments could also (ii) expand our knowledge regarding the development of the VM DA neurons. By understanding the roles of CHL1 in VM DA development, we could use this knowledge to enhance the generation of VM DA neurons from human PSCs. Additionally, CHL1 could be utilized to enhance the outcomes of cellular replacement therapies for different neurological disorders, such as Parkinson’s disease.
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ItemUnderstanding the regulation of adult-neurogenesis following acute and neurodegenerative brain injury( 2016)Adult-neurogenesis is a physiological event occurring under normal conditions, which produces new neurons in specific brain regions and may have significant potential as a target for brain repair. Neurogenesis is impaired in certain neurological conditions including neurodegenerative disorders like Parkinson's disease or acute injuries like stroke. In the last 50 years, tremendous effort has been put into understanding adult neurogenesis and how it is affected and can be manipulated in disease, with the hope to develop regenerative approaches for brain-repair. However, while extensive progress has been made, the clear mechanisms underlying its regulation remain to be further characterised. The focus of this thesis is to comprehend the potential and to define the limitations for applications related to neuronal replacement therapies based on ‘endogenous repair’. We investigated two different disease models: progressive injury, Parkinson’s disease and an acute injury, stroke. In a rat model of Parkinson’s disease (PD), we investigated the relative importance of the dopaminergic and noradrenergic systems in regulation of hippocampal neurogenesis. We demonstrated that, contrary to existing literature, the dopaminergic system does not project directly to the neurogenic dentate gyrus, which receives a rich noradrenergic innervation. Although studies have concluded that dopaminergic projection to the dentate gyrus regulates hippocampal neurogenesis, we further showed that the depletion of these two systems had no impact on hippocampal neurogenesis, suggesting mechanisms independent of dopamine and noradrenaline loss may underlie reduced neurogenesis in PD. Furthermore, we presented a new tool for the study of noradrenergic neuronal transplantation, the DBH-eGFP reporter mouse. To assess the regenerative capacity of the brain after an acute injury, we used a model of ischemia using the vasoconstrictor endothelin-1 (ET-1). The results contribute to a mixed literature where there has been conjecture as to whether the adult brain is capable of generation region-specific neuronal phenotype in response to injury. We found that after ischemic injury to the striatum of adult rats, the proliferative response does not include the production of new projection neurons. Interestingly, we also found that even in the early postnatal brain, where striatal projection neurons are being actively generated, stroke does not impact on the rate of neuronal production. This gives cause to rethink the brain's capacity for self-repair. This thesis comes at a time where interest for the development of regenerative medicine is very high and where the use of endogenous stem cells as a therapeutic approach is very attractive. The work presented here showed that while the brain may have potential for self-repair, there are clear limitations in its regenerative potential and the use of endogenous stem cells for therapeutics is still a long way away
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ItemReal time modelling of neuronal excitability using sodium channel dynamic-clamp( 2016)Voltage-gated sodium channels are responsible for the generation of action potentials in excitable cells. In the central nervous system they are critical for regulating network excitability, as is evident from the pathological consequences of sodium channel dysfunction. Over the past two decades more than 700 mutations among the genes that encode sodium channels have been identified in patients with epilepsy. Further, many first line anti-epileptic drugs have been shown to exert their influence over the nervous system through modulating sodium channel behaviour. This speaks to the importance of studying sodium channels to develop a better understanding, and more effective treatments for epilepsy. Common methods for characterising changes in sodium channel function either use a reduced preparation where sodium currents are recorded in isolation, or an intact, complex systems – such as animal models – where changes in neuronal or network excitability are measured. These dispirit approaches indicate the need for a method that can reconcile changes in sodium channel biophysics with changes in neuronal excitability. To address this problem we present a method that couples real sodium channels with a computer model of a neuronal compartment, so that the impact of channel behaviour on membrane dynamics can immediately be determined. To validate this system we present a case study looking at the interaction of the well-established antiepileptic carbamazepine, with the NaV1.6 sodium channel. Initially we performed conventional voltage-clamp experiments to quantify the effect of carbamazepine on NaV1.6 channels stably expressed in Chinese Hamster Ovary (CHO) cells. This study demonstrated that carbamazepine block of NaV1.6 channels is increased with membrane depolarisation and that the drug most likely binds channels in the fast-inactivated state, and then accelerates entry into a slow-inactivated state. Informed by the results of this voltage-clamp study we attempted to construct a Markov kinetic model to validate the hypothesised mechanism of carbamazepine interaction with NaV1.6 channels. While the model failed to recapitulate voltage-clamp data, we were able to construct an alternative model that could phenomenologically describe our data but was not mechanistically accurate. After demonstrating the challenge of modelling drug-channel interaction using traditional methods we developed a dynamic-clamp implementation merging the voltage-clamped NaV1.6 channels with a biophysically realistic model of the neuronal axon initial segment. Using this method, we could recapitulate results from previously published studies of carbamazepine action on intact neurons. We then extended the technique to emulate in vivo neuronal dynamics by incorporating a description of synaptic background noise into the compartment model. This provided novel insight into how carbamazepine interaction with NaV1.6 channels could alter neuronal excitability, revealing that it chronically lowered channel availability under physiological and seizure-like conditions. This global inhibition could supress neuronal activity and account for some cognitive side-effects reported by patients. The findings generated by the dynamic-clamp analysis of carbamazepine action on NaV1.6 channels demonstrate the potential of this method to directly relate changes in sodium channel function to changes in neuronal excitability – this information is crucial to understanding how seizures can both emerge and be controlled.
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ItemThe role of CRFR1 in addiction and anxiety disorders( 2016)Addiction and anxiety disorders are highly co-morbid, and represent a huge burden on society. The central role of stress-reactivity in the pathogenesis and maintenance of both of these diseases has led to the identification of corticotropin-releasing factor (CRF) signalling as a key factor in these effects. The focus of this thesis was the ventral tegmental area (VTA), as it is a site where reward- and fear- related circuitry converge and can be modulated by CRF. The broad aims of this thesis were to examine the role of VTA CRF receptor 1 (CRFR1) in animal models of reward-seeking and conditioned fear to understand how these systems can become dysregulated in addiction and anxiety disorders. To this end, chapter 3 of this thesis validated a technique for the viral-mediated downregulation of CRFR1 within the VTA, and chapter 4 established a novel model of stress-induced reinstatement of cocaine-seeking in mice. These techniques were then implemented to examine the effects of VTA CRFR1 knockdown on the acquisition, extinction, and reinstatement behaviours. Chapters 5 and 6 are two separate publications demonstrating that VTA CRFR1 signalling is differentially involved in various components of cocaine-seeking and conditioned fear. In chapter 5, knockdown of CRFR1 in the VTA blocked stress-induced reinstatement of cocaine-seeking and attenuated cued cocaine-seeking, without any effects on self-administration or extinction responding. This was a specific effect on drug-related behaviours, as there were no changes to operant responding for sucrose rewards. In chapter 6, VTA CRFR1 knockdown enhanced the expression of conditioned fear, but had no effects on fear extinction or reinstatement. This evidence suggests that CRFR1 participates in distinct subcircuits within the VTA which mediate fear and reward-seeking.
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ItemEngineered exosomes as carriers of biologically active proteins( 2016)A critical component for devising drug delivery systems is to package and protect molecules with the ability to release them specifically in a temporal and spatial manner. To date a number of systems have been developed as drug delivery vehicles; however, many have limitations in regard to immunogenicity, packaging efficiency and stability. Recent interest in vectors has shifted to naturally occurring nanovesicles called exosomes which can package proteins, lipids, DNA and various forms of RNA for delivery to recipient cells. These small vesicles are released from nearly every cell type and are known to play a role in cellular communication pathways through the delivery of the aforementioned cargo. However, mechanisms for loading functional molecules into exosomes are relatively unexplored. Here I report the use of the evolutionarily conserved late-domain (L-domain) pathway as a mechanism for loading exogenous proteins into exosomes. I demonstrate that labelling of a target protein, Cre recombinase, with a WW-tag leads to recognition by the L-domain protein Ndfip1, resulting in ubiquitination and loading into exosomes. My results show that Ndfip1 expression acts as a molecular switch for exosomal packaging of WW-Cre that can be suppressed using the exosome inhibitor GW4869. When taken up by floxed reporter cells, exosomes containing WW-Cre were capable of inducing DNA recombination indicating functional delivery of the protein to recipient cells. Using this functional in vitro assay I was able to identify differences in the communication potential between distinct cell types as well as the functionality of exosomes after storage. Engineered exosomes were administered to the brain of transgenic reporter mice using the nasal route to test for intracellular protein delivery in vivo. This resulted in the transport of engineered exosomes predominantly to recipient neurons in a number of brain regions. The ability to engineer exosomes to deliver biologically active proteins across the blood-brain barrier represents an important step for the development of therapeutics to treat brain diseases.
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ItemThe BDNF Val66Met single nucleotide polymorphism alters behaviour via sensitivity to glucocorticoid signalling( 2016)Brain-Derived Neurotrophic Factor (BDNF) plays a critical role in supporting neuronal survival, growth, differentiation and plasticity, and has been implicated in a wide variety of psychiatric disorders. A common coding polymorphism, termed BDNF Val66Met due to a single amino acid substitution of Valine→Methionine at codon 66 within the BDNF prodomain, has been associated with anxiety, affective, eating, mood and psychotic disorders as well as suicidal behaviour. However, clinical investigations have reported inconsistent data between studies, suggesting that this polymorphism is unlikely to adhere to a simple genetic model. Given evidence that carriers of the BDNF Val66Met polymorphism are more vulnerable to the effects of stress, are more likely to experience stressful life events and are at a heightened risk of stress-related illness, I hypothesised that glucocorticoids may interact with BDNF Val66Met genotype to alter behavioural and molecular endophenotypes of anxiety, mood and psychotic disorders. To explore this hypothesis, a novel transgenic mouse line was utilised that was genetically modified to carry the BDNF Val66Met polymorphism as well as an extended sequence that humanizes the coding region of the mouse Bdnf gene. These modifications result in the expression of a humanized BDNF coding transcript in vivo via endogenous mouse promoters. To model stress, a chronic corticosterone (CORT) exposure paradigm was adapted. Mice were chronically treated with a 25mg/L dose of chronic CORT during late-adolescence (weeks 6-9), before undergoing behavioural phenotyping in adulthood (weeks 11-14). Following sacrifice, brains were collected and utilised for molecular profiling of neurotrophin signalling and stress-sensitivity markers in the dorsal hippocampus (DHP), ventral hippocampus (VHP) and medial prefrontal cortex (mPFC). The principal result of this experimentation was the recapitulation of hippocampus-dependent memory deficits, which were assessed using the Y-maze and a contextual fear conditioning paradigm, in hBDNF Met/Met mice as has been observed in human carriers of this polymorphism. Interestingly, these memory deficits were ‘rescued’ by chronic CORT treatment. Increased glucocorticoid receptor (GR) expression was observed in the DHP of adolescent hBDNF Met/Met mice and VHP of adult hBDNF Met/Met mice. Interestingly, chronic CORT treatment normalized GR expression in the VHP of adult hBDNF Met/Met mice, reinstating GR expression values in CORT-treated hBDNF Met/Met mice to levels consistent with hBDNF Val/Val controls. On the forced-swim test, hBDNF Met/Met mice exhibited increased behavioural despair at baseline. However, following chronic CORT a convergent phenotype emerged amongst hBDNF Val/Val control mice. This behaviour inversely coincided with tyrosine hydroxylase expression in the mPFC of both genotype groups, implicating that catecholamine innervation of this brain region is altered in hBDNF Met/Met mice at baseline and in hBDNF Val/Val wildtype mice as a consequence of allostatic overload. Lastly, a ‘heterozygote disadvantage’ phenotype was observed for prepulse inhibition (PPI). Specifically, hBDNF Val/Met mice had deficient PPI relative to hBDNF Val/Val mice at the commonly used 100msec PPI interstimulus interval and, following CORT treatment, worse PPI than both hBDNF Val/Val and hBDNF Met/Met mice at the shorter 30msec interstimulus interval. These data suggest that there is an alteration in the PPI circuitry of hBDNF Val/Met heterozygote mice that is not present in either hBDNF Val/Val or hBDNF Met/Met homozygote groups. Cumulatively, these experiments support the hypothesis that the BDNF Val66Met polymorphism alters sensitivity to glucocorticoids, and that this vulnerability can lead to divergent behavioural outcomes in hBDNF Val/Val, hBDNF Val/Met and hBDNF Met/Met mice in a gene-dosage independent manner.
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ItemNew insights into molecular and cellular pathways of neurodegeneration in amyotrophic lateral sclerosis models( 2016)Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and paralysing neurological disorder usually fatal within 2-5 years from diagnosis. First described by Jean-Martin Charcot in the late 1860s, ALS still remains a terminal disease with no effective treatments or cure. Riluzole is the only clinically-approved drug for ALS that may extend survival by 2-3 months. Therefore, there is an urgent need to understand the underlying pathogenesis of ALS to better guide development of disease-modifying treatment strategies. This thesis investigates the molecular basis of three inter-related pathogenic mechanisms implicated in motor neuron vulnerability and loss in ALS: defective energy homeostasis; disruption of protein homeostasis and abnormal RNA homeostasis. Two leading mouse models of ALS were implemented in these studies; transgenic SOD1G93A and TDP-43A315T mice in which novel pharmacological and genetic interventions were evaluated for efficacy. To examine whether defective energy metabolism is causal or consequential in the pathological cascade of ALS, the role of the key metabolic and stress sensor; AMP-activated protein kinase (AMPK) was investigated for the first time in two ALS mouse models. AMPK activation in the spinal cord associated with symptom progression, but not onset, in SOD1G93A mice, implicating AMPK activity in mediating disease course. Conversely, AMPK inactivation occurred in spinal cord and brain of pre-symptomatic TDP-43A315T mice by a protein phosphatase 2A-dependent mechanism, identifying a novel regulation of AMPK activity by pathogenic TDP-43. AMPK inactivity may therefore drive disease initiation in this mouse model. Hence, mutant SOD1 and TDP-43 exert contrasting effects on regulation of AMPK activation which may reflect intrinsic differences in energy metabolism and neurodegeneration in these two ALS mouse models. Next, a novel pharmacological strategy to improve protein homeostasis and motor neuron health was developed and evaluated for ALS. The intracellular catabolic pathway, autophagy, particularly macroautophagy, was robustly induced in mutant SOD1 and TDP-43 models of ALS. To potentiate autophagy in ALS, a novel autophagy enhancer rilmenidine was used to stimulate mTOR-independent macroautophagy in mutant SOD1 cell and mouse models. Rilmenidine treatment achieved efficient macroautophagy induction in vitro and in vivo. However, the treatment worsened motor neuron degeneration and survival of male SOD1G93A mice by exacerbating accumulation of insoluble and misfolded SOD1 species and aggregates in spinal cords. Thus, macroautophagy stimulation using rilmenidine may mediate disease progression in this specific mouse model of ALS. Lastly, a new gene therapy strategy to alleviate defects in the RNA binding protein TDP-43 was investigated. Survival motor neuron (SMN) protein deficiency causes progressive motor neuron degeneration in spinal muscular atrophy (SMA) and may be linked to pathology in ALS. SMN overexpression was previously determined to be beneficial in mutant SOD1 models of ALS. To extend these studies to TDP-43 proteinopathy, upregulation and accumulation of endogenous SMN protein into stress granules within motor neurons was demonstrated for the first time in TDP-43A315T mice. The impact of forced SMN overexpression in TDP-43A315T mice was examined, revealing improved SMN nuclear targeting, motor neuron survival, neuroinflammation and metabolic deficits as shown by AMPK activation, in female mice. Furthermore, levels of androgen receptor (AR), mutations of which cause spinal bulbar muscular atrophy (SBMA), were significantly impaired in spinal cords of male TDP-43A315T mice. This provides evidence for shared biochemical pathways in ALS, SMA and SBMA, mediated by deficiency of factors such as SMN and AR which confer motor neuron vulnerability. In summary, in mutant SOD1-linked disease, persistent AMPK signalling and autophagy activation in motor neurons may be key determinants of disease progression. In mutant TDP-43-mediated ALS, AMPK inactivation and cytoplasmic accumulation of SMN in motor neurons may be early events triggering disease onset. In conclusion, this thesis provides novel insights into pathogenic mechanisms underlying disruption of energy, protein and RNA homeostasis within motor neurons and significant clues to therapeutic alleviation of these defective pathways in ALS. In addition, this thesis identifies new links between three main neurological disorders affecting the motor system of humans; ALS, SMA and SBMA, mediated by dysregulation of SMN and AR, suggesting shared pathogenic pathways. Finally, this work importantly extends the spectrum of motor neuron diseases that may benefit from SMN restoration, excitingly paving the way for future therapeutic development and testing of SMN enhancing agents for ALS.