Anatomy and Neuroscience - Theses
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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.
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ItemMolecular and cellular insights into mitochondrial contributions to neuronal autophagy: links to energetics and mitophagy( 2016)Neurones are essential for brain homeostasis and as highly metabolic cells rely on mitochondrial oxidative phosphorylation (OXPHOS) for energy. The integrity and functionality of mitochondria are critical for neuronal survival, and the involvement of dysfunctional mitochondria is recognized as a common theme amongst various neuropathologies. New evidence has suggested that the inappropriate clearance of dysfunctional mitochondria via autophagy (termed mitophagy) determines the pathogenesis of neurodegenerative diseases such as Parkinson’s disease. Mechanistic studies of mitophagy have been undertaken using mammalian cell lines, but this research lacks relevance to neuropathologies. This thesis investigates triggers of autophagy/mitophagy in primary neurones, and specifically if disruption of mitochondrial bioenergetics triggers neuronal autophagy, and mitophagy in particular. Cultures of primary cerebellar granule cells (CGCs) were utilized and inhibitors of the OXPHOS complexes (rotenone, 3-Nitropropionic acid, antimycin A, potassium cyanide and oligomycin targeting complex I-V, respectively), were employed to induce bioenergetic dysfunction of mitochondria. Initial investigations using MTT cell viability assay, phase contrast microscopy and cellular membrane permeabilization detected by propidium iodide staining, determined appropriate concentrations of OXPHOS inhibitors which induced effective mitochondrial damage producing slow neuronal degeneration. From this baseline adverse effects of OXPHOS inhibitors on mitochondrial bioenergetics were documented by monitoring reductions in cellular ATP level, mitochondrial membrane potential (ΔΨm) and oxygen consumption rate (OCR) of CGCs. ΔΨm was rapidly dissipated in CGCs exposed to the inhibitors of complexes I, III and IV (rotenone, antimycin A and potassium cyanide, respectively), whilst the inhibitor of complex II, 3- Nitropropionic acid, produced a much slower reduction of ΔΨm. Employing Seahorse XF24 technology allowed an incisive readout of mitochondrial functional changes where significant bioenergetic impairment was observed subsequent to inhibition of complexes I and II, which are core components of energy metabolism regulating the redox balance (NAD+/NADH levels) and TCA cycle. Existent evidence indicates depolarization of ΔΨm triggers mitophagy in mammalian cell lines, however CGCs display contrasting results where ΔΨm depolarization via complex III and IV inhibition was insufficient to elicit mitophagy despite the inducer of mitophagy, PINK1, being mobilized to mitochondria. In contrast, inhibition of complexes I and II induced mitophagy, as indicated by PINK1 mobilization and disappearance of the pH-sensitive fluorescence mitophagy reporter, mt-Rosella. Western immunoblotting of the general autophagy marker, LC3, and monitoring of acidic vesicles with monodansylcadaverine revealed activation of autophagic flux in CGCs exposed to inhibitors of complexes I-IV, indicating general autophagy in response to bioenergetic impairment irrespective of mitophagy induction. Results presented herein reveal the complexity of neuronal mitophagy and that ΔΨm may not be a necessary trigger of neuronal mitophagy. Thus inhibition of individual respiratory complexes, and notably complexes I and II, may underlie the triggering of mitophagy in primary neurones, where different mechanisms induce mitophagy in neurones compared to immortalized cell lines. This difference may be due to the unique bioenergetic dependence of neurones. Understanding the mechanisms of mitophagy and autophagy in primary neurones provides valuable insights into therapeutic approaches for neurodegenerative diseases.
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ItemOn type-1 interferons, neuro-inflammation and Alzheimer's disease( 2015)Alzheimer’s disease (AD) is the most common form of dementia worldwide. Hyper-phosphorylation of tau, leading to intracellular neurofibrillary tangles, and accumulation of the amyloid-β (Aβ) peptide, leading to formation extracellular plaques, are the two key brain proteinopathies associated with the progressive neuro-degenerative disease. To date, therapeutics targeting these pathologies have proven ineffective in the treatment of AD and highlights the need for new lines of investigation into disease mechanisms. Neuro-inflammation is evident in AD patients, comprising of enhanced gliosis surrounding Aβ deposits and pro-inflammatory cytokine load. This dysregulated innate inflammatory response is deleterious and facilitates neuro-degeneration. Identifying critical mediators controlling this neuro-inflammation will prove beneficial in developing anti-inflammatory therapies for the treatment of AD. The type-1 interferons (IFNs) are pleiotropic cytokines that control pro-inflammatory cytokine secretion and are master regulators of the innate immune response. This thesis carries the hypothesis that the type-1 IFNs play a critical role in the exacerbation of neuro-inflammation and actively contribute to the progression of AD. This thesis aimed to characterise the role of type-1 IFNs in the neuro-inflammatory response to soluble Aβ1-42 in CNS cell types, evaluate the effect of removing type-1 IFN signalling in the APPSWE/PS1ΔE9 mouse model of AD and hence identify a role for type-1 IFNs in the progression of AD. Soluble Aβ1-42 triggers a type-1 IFN neuro-inflammatory response in primary cultured neurons. Removal of type-1 IFN signalling (IFNAR1-/-) in these cultures attenuated pro-inflammatory responses to Aβ1-42 affording protection against neurotoxicity via attenuation of pro-apoptotic caspase-3 activation. The use of Myd88-/-, IRF7 and IRF3 knockdown cultures, critical in toll-like receptor-dependent signalling, identified that neurons utilise this receptor family to detect Aβ1-42 and initiate a neuro-degenerative type-1 IFN response. Primary IFNAR1-/- mixed astrocyte and microglial cultures display an attenuated type-1 IFN and pro-inflammatory cytokine response to Aβ1-42. These cultures adopt an anti-inflammatory and neuro-protective M2-like polarisation state, opposing the wildtype neuro-degenerative M1-like response to soluble Aβ1-42. To investigate the role of in vivo type-1 IFN signalling in the progression of AD, APPSWE/PS1ΔE9 mice lacking type-1 IFN signaling were generated. APPSWE/PS1ΔE9 x IFNAR1-/- were partially rescued from spatial learning and memory deficits as assessed by the Morris water maze. These mice displayed no alterations in amyloid plaque load but reduced soluble Aβ monomer concentrations were detected. The type-1 IFN response was attenuated in APPSWE/PS1ΔE9 x IFNAR1-/- mice displaying altered pro-inflammatory cytokine expression. Interestingly, cortical astrogliosis was elevated in these mice but microgliosis was attenuated. These microglial populations adopted a neuro-protective M2-like activation state, supporting our in vitro findings. Finally, a type-1 IFN signature was evident in the brains of human post-mortem AD patients Findings from this thesis identify the type-1 IFNs as key mediators of the neuro-inflammatory response in AD. This response is deleterious to disease progression and suggests that targeting type-1 IFN signalling may be therapeutically relevant for anti-inflammatory treatment of AD.
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ItemThe effects of stress on the onset and progression of Huntington's disease in a transgenic mouse model( 2014)Huntington’s disease (HD) is a neurodegenerative disorder largely governed by genetics. The cause of the disease is a fully penetrant gene mutation, inherited by autosomal dominant transmission. The length of this mutation also predicts the age of disease onset, which can range from childhood to late adulthood. Work from our lab on the R6/1 transgenic mouse model of HD was the first to show that environmental factors can alter symptom progression. Environmental enrichment and voluntary wheel running delayed or ameliorated the triad of motor, affective and cognitive dysfunctions in HD mice. Recent clinical studies also suggest that lifestyle factors can affect the age of onset. Currently, there are no treatments to slow or change the course of HD so environmental interventions may offer a feasible approach to extend the symptom-free years in HD gene-positive individuals. There is evidence to suggest that the stress response is abnormal in HD mice and patients. The present study is the first to investigate the impact of stressors on the onset and progression in an animal model of HD. We used an acute (Chapter 3) and two chronic stress paradigms (Chapters 4 and 6) to assess the impact on characteristic symptoms of HD. We also extended the phenotyping of R6/1 HD mice to include behaviours of ethological relevance (Chapter 5). All 3 stress protocols were able modify various functions in R6/1 HD mice, notably accelerating cognitive decline and further impairing olfactory deficits. This work contributes data for sex differences in the HD phenotype and to the general stress literature. Importantly, we show that stress is not only able to modulate specific behaviours in HD mice, but that the gene mutation may confer a susceptibility to the negative effects of stress. Therefore, behavioural management therapy in combination with other lifestyle changes may help manage the course of the disease in gene positive individuals.
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ItemTesting paraclinical outcome markers in optic neuritis and a study of the phenomenology and treatment of tremor in multiple sclerosis( 2012)Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterised by multi-focal inflammatory demyelinating plaques and significant neuroaxonal degeneration. The clinical presentation and disease course of MS is heterogeneous and ranges from clinically isolated syndromes such as optic neuritis (ON), to disabling progressive disease at which stage tremor can be a feature. This heterogeneity reflects the multi-focal nature of the CNS injuries and suggests that the relationships between neuronal injury and clinical presentations are complex. Focused clinical and paraclinical assessments of defined neural pathways associated with discrete clinical presentations can help improve our understanding of complex clinico-pathological relationships. This thesis examined clinical manifestations and outcomes in two models at the opposite ends of the MS clinical spectrum namely acute isolated ON and MS-related tremor. The aims of section one was to comprehensively study changes in clinical and paraclinical markers after ON and more specifically to evaluate the ability of these markers to serve as predictive biomarkers of clinical and axonal outcomes after ON. The second part of this thesis aimed to perform a detailed clinical phenomenology study in MS tremor patients to help define possible underlying neuroanatomical networks and broaden understanding of injury in secondary progressive MS in general. In addition, the efficacy and safety of onabotulinumtoxin-a in the treatment of MS tremor was evaluated. In Section one, forty patients with acute, idiopathic, unilateral optic neuritis with early MS or at high risk of MS were recruited. Patients were studied prospectively over 12 months at multiple time-points. Ten healthy age and sex-matched controls were tested twice to inspect inter-scan and inter-subject variability. All participants had a detailed neurological assessment, visual acuity testing (high and low-contrast and colour vision), optic nerve diffusion tensor imaging (DTI), multi-focal visual evoked potential (mfVEP), and optical coherence tomography (OCT). The main outcome measures were 2.5% low contrast visual acuity, mfVEP amplitude and retinal nerve fibre layer thickness (RNFL) at 12 months after ON. Significant changes in clinical and paraclinical measures after ON, particularly in the first 3 months were demonstrated. Despite some initial recovery, persistent visual, functional and neuroaxonal deficits persisted at 12 months. Multivariate models identified decreased high contrast visual acuity (logMAR notation) at 1 month after ON to be the earliest predictor of persistent visual loss at 12 months. Decreased 3-month colour vision and RNFL thickness also both significantly predicted poor visual recovery. The earliest predictors of axonal outcomes at 12 months after ON were decreased 1-month mfVEP amplitude, low contrast acuity and optic nerve axial diffusivity. Decreased optic nerve axial diffusivity was the best predictor of severe combined (persistent mfVEP amplitude loss and/or RNFL thinning of greater than 30%) axonal loss. Both mfVEP amplitude and clinical assessments are limited in the hyper- acute setting due to inflammatory oedema, conduction block and variable patient performance. Therefore, the finding of an early structural marker, optic nerve axial diffusivity, that is able to predict axonal outcome after ON is important as this measure could serve as a biomarker in future studies of putative neuroprotective therapies by identifying those patients at most risk of permanent axonal loss. In Section 2 of the thesis, a cross-sectional study of 54 MS patients with and without tremor was undertaken. Of the 27 tremor cases, 25 were included in a double-blind randomized placebo-controlled crossover trial (RCT) to evaluate the efficacy of onabotulinumtoxin-a. A detailed comparison between MS patients with and without tremor was completed. Patients underwent a detailed history and neurological assessment that included the Expanded Disability Severity Scale (EDSS) as well assessments of cerebellar ataxia, dystonia and tremor-related quality of life. All patients were assessed for the presence of tremor and tremor severity was rated using the Bain score, a validated MS tremor severity score to rate tremor severity, writing and drawing an Archimedes spiral. Functional tasks such as writing, drawing, drinking from a cup and pouring water from one cup to another were completed. A blinded, independent observer, rated standardized video assessments. In the RCT, patients were assessed 6 weekly for 6 months with baseline and 3 months injections of either onabotulinumtoxin- a or placebo. MS tremor was found to be predominantly an action tremor of the upper limb with prominent cerebellar and dystonic features. Dystonic features (including geste antagonistes, mirror movements, dystonic posturing and writer’s cramp) were more prominent and more severe in patients with tremor than those without. A further novel finding was the impact of dystonia severity in the affected limb on tremor severity. This result highlights the complexity of MS tremor as a movement disorder with injury that is not localized to the cerebellum alone, but rather to the wider cerebello-thalamo-cortical, pallido-thalamic as well as cortico-cerebellar network. In a RCT of the efficacy of onabotulinumtoxin-a in MS tremor, the drug was found to reduce MS tremor severity by an average of 40%, with writing and drawing severity improving by an average of 30%. Injections were well tolerated with transient focal weakness being the predominant side effect. The efficacy of onabotulinumtoxin-a likely reflects modulation of the central tremor-generating networks by modification of peripheral stretch reflexes and muscle- spindle excitability. In addition, the efficacy of a drug used predominantly for focal dystonia underscores the importance of recognizing dystonia as a feature of MS tremor.