Florey Department of Neuroscience and Mental Health - Theses
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ItemAn investigation into the toxic mechanism of alpha-synuclein: implications for Parkinson’s disease therapeutics( 2018)Parkinson’s disease is a neurodegenerative disorder that causes motor, cognitive and neuropsychiatric deficits in patients. Current therapies do not stop ongoing neurodegeneration, and investigating biochemical features present in brain regions affected by cell death may enable identification of mediators of neurodegeneration to identify targets for potential disease-modifying drugs. α-synuclein is a protein found aggregated in Lewy bodies, a major pathology present in patients’ brains, and mutations and variants in the α-synuclein-encoding gene can increase susceptibility to Parkinson’s disease. Observations from human post-mortem studies fuelled a hypothesis that α-synuclein pathology may spread between adjacent brain regions and contribute to neurodegeneration, which were further supported by animal studies utilising injection of aggregated α-synuclein into the brain. The purpose of this thesis was to optimise a cell culture model to test the mechanism of α-synuclein toxicity. In multiple cell types, I applied aggregated α-synuclein pre-formed fibrils (PFFs), to model toxic pathways activated by pathological α-synuclein. Since iron levels are elevated in patients’ brains, and improvement in patient’s motor function was observed in response to iron chelation therapy in a phase II clinical trial, I hypothesised that α-synuclein PFFs might cause toxicity via ferroptosis, an iron- and lipid peroxidation-dependent cell death pathway identified in 2012 that warrants investigation as a cause of cell death in Parkinson’s disease. I addressed this hypothesis using a predominantly pharmacological approach in which drugs known to inhibit or exacerbate ferroptosis were added to cells prior to α-synuclein PFFs, and the effect of each drug on toxicity was compared to that of ferroptosis inducer, erastin. Toxicity of α-synuclein was inhibited by liproxstatin-1, a classic anti-ferroptotic agent, but was otherwise not impacted by a range of other ferroptosis inhibitors or inducers. Lipid peroxide levels, which elevate during ferroptosis, were not changed following administration of α-synuclein PFFs, indicating that liproxstatin-1 likely reduced α-synuclein toxicity via a ferroptosis-independent mechanism. Liproxstatin-1 concentrations that were protective against α-synuclein toxicity caused similar elevation of autophagy-lysosomal pathway markers as known lysosomal acidification inhibitors, chloroquine and bafilomycin A1. This highlighted lysosomal inhibition as a potential mechanism by which α-synuclein PFF toxicity might be reduced. Both chloroquine and bafilomycin, and inhibitors of lysosomal cysteine proteases, leupeptin and E-64D, significantly recovered loss of viability in α-synuclein-treated cells. In parallel confocal imaging analysis, I observed that while the majority of α-synuclein remained outside of the cell, a small proportion entered the endo-lysosomal pathway; furthermore, chloroquine treatment did not prevent PFF association with this pathway. In contrast, heparin also protected against α-synuclein toxicity, and prevented membrane association and cellular internalisation of α-synuclein PFFs. I concluded that the toxicity of α-synuclein was likely caused by internalised α-synuclein associated with the lysosome, with heparin reducing toxicity by interfering with α-synuclein uptake. The protection conferred by lysosomal inhibitors may not involve preventing α-synuclein uptake into the lysosome but suggested that aggregated α-synuclein requires a functional lysosome to initiate toxicity. Thus preventing aggregated α-synuclein from entering cells, or inhibiting a lysosomal function critical for its toxicity should be an aim for future Parkinson’s disease therapies.
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ItemSpecific biomarkers for acute stroke: comparison between human and animal models and development of a new way to improve acute stroke conditions( 2018)Stroke is the third most common cause of death in most Western countries and the major cause of disability. The distinction between stroke subtypes and knowledge of the time of stroke onset is critical in clinical practice. The most specific and biologically powerful treatment for acute ischaemic stroke is thrombolysis with recombinant tissue plasminogen activator (rt-PA) given within the first 4.5 hours of ischaemic stroke onset but this therapy is disappointingly underused. This mainly because of unknown symptoms onset time and uncertainty about stroke diagnostic. Neuroimaging can help decide who and how to treat. Nevertheless, neuroimaging is expensive, has contra-indications and is not always readily available. Cheap and easily measured blood biomarkers serve similar roles for other diseases. The traditional approach to stroke biomarker discovery has been to select candidate markers based on their known involvement in the stroke pathophysiology. This “pick your best candidate” approach inevitably means selection from only a small pool of what is theoretically available. High throughput technologies such as whole genome microarrays and proteomics permit unbiased selection of molecular markers by examining all of genes and proteins expressed in a tissue. In this thesis, these agnostic approaches were used to identify characteristic blood RNA and protein expression profiles occurring after stroke. In the first chapters, we show that most acute stroke patients do not have the indicated imaging done in an appropriate time window, therefore blood biomarkers would have a useful niche. We also show that, to date, most candidate blood biomarkers have not found use in the clinic because they have been selected from experiments that do not address the rapidly changing nature of stroke and have been identified by an ad hoc process that only scratches the surface of the potential candidates available. A pilot gene array experiment performed in rats to specifically look for hyper-acute changes that might be clinically informative in a way not easily realizable in humans, identified gene expression changes of great amplitude which changed markedly with time. This indicated that it should be possible to generate a stroke clock for use in the clinic. A larger follow up confirmation experiment designed also to examine the influence of experimental comorbidities demonstrated that many of the changes could be attributed to the surgical intervention needed to induce stroke in rats. This has important implications for our understanding of stroke inflammatory biology and also suggests the need for a different approach to stroke biomarker discovery. Examination of protein expression in bloods from a clinical trial of hypothermia identified new candidate biomarkers. A clinical trial to examine blood RNA expression, ultimately performed as a pilot because of recruitment difficulties, was designed to examine temporal change. It confirmed that collection of sequential blood samples in a clinically relevant time frame is possible and identified time dependent gene expression with overlap with the rat data. This indicates that generation of a clinically useful stroke clock should indeed be possible.
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ItemSpatial and temporal surveillance of the mechanisms controlling proteome foldedness via a FRET-based biosensor( 2018)Proteostasis (protein homeostasis) is essential for keeping the proteome functional. This process controls protein synthesis, folding and degradation and involves hundreds of genes, including those encoding chaperones, to form extensive quality control (QC) networks (Kim et al., 2013). Imbalances in proteostasis are implicated in a range of aggregation-based neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS), Huntington’s and Alzheimer’s diseases (Morimoto et al., 2014; Vilchez et al., 2014). Currently there is a lack of capacity to quantitatively measure proteostasis imbalance and therefore we are limited in understanding how proteostasis imbalance manifests during disease. A new biosensor system has been developed by our lab to address this shortfall. The biosensor is a genetically encoded unfolded “bait” flanked by two fluorescent proteins to assay foldedness by fluorescence resonance energy transfer (FRET). Proteostasis efficiency is reported by measurement of the efficiency to which the bait interacts with the QC network. In this master’s project, the biosensor was further targeted to organelles to allow for a higher degree of spatiotemporal control. Signalling peptides were used to target the biosensor to specialised microenvironments, and successful targeting was achieved in the Golgi apparatus and nucleus. Investigations into nuclear proteostasis revealed the biosensor behaved predictably to chaperone overexpression (Hsp40 and Hsp70 co-expression) or inhibition (Hsp70 or Hsp90 inhibition). Polyglutamine (PolyQ) expansions of non-pathogenic (Q25) to pathogenic (Q72) lengths reduced the biosensor foldedness and decreased aggregation, which is consistent with an increase in chaperone supply. The biosensor was also adapted to express in the body wall muscles of Caenorhabditis elegans to examine change in proteostasis across age and in an organismal context. The biosensor was successfully expressed in the model organism, with potential sub-microscopic and variant biosensor expression level confounding data analysis. The C. elegans reporter lines were successfully crossed with lines expressing Aβ (1-42) demonstrating the ability of the biosensor to report on disease states. Moving forward, the generation of low-expression, single-copy C. elegans biosensor lines would allow for steady, matched expression and enhanced capacity for comparison between worm lines.
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ItemUnderstanding the role of ATP13A2 in endo-lysosomal pathways( 2018)Parkinson’s disease (PD) is a common neurodegenerative disease affecting 1 to 2% people aged over 65. Cytoplasmic inclusions are pathognomonic of PD and point to dysregulation of protein clearance as a central part of the pathology. Two forms of familial PD result from mutations in the genes for the ATP13A2 and α-synuclein proteins. One of these proteins (α-synuclein) forms the characteristic inclusions of PD and is thus likely to be affected by dysregulated protein clearance, whereas the other is part of the endo-lysosomal system (ELS), which is a central component of protein clearance. The predictions on which these studies were based on were that removal of ATP13A2 would have i) a deleterious effect on ELS function; ii) induce aspects of neurodegeneration; iii) dopaminergic systems would be most susceptible; iv) the presence of high levels of α-synuclein would exacerbate these changes. None of these proved correct in these mice and instead it appears that removal of ATP13A2 results in i) improved capacity of ELS function; ii) there is no loss of neurons but there is neuro gliosis; iii) there is plasticity in the dopaminergic systems that suggest a response to altered neurotransmission; iv) the presence of high levels of α-synuclein did not exacerbate any aspect of the pathology and the absence of ATP13A2 in the presence of increased α-synuclein expression resulted in improved clearance of α-synuclein. These unexpected findings open up several new avenues for investigation of the normal physiological role of these proteins in the ELS and also for investigating whether understanding the mechanisms of the improved α-synuclein clearance might reveal new therapeutic targets for protein clearance disorders.
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ItemBiochemical mechanisms of biomineralization and elemental incorporation in otoliths: implications for fish and fisheries research( 2018)All vertebrates have small bioinorganic “earstones” in their inner ear labyrinth that are essential for hearing and balance. While otoliths play a vital anatomical role in fish, their true value to science is as biochronometers, largely due to their unique pattern of growth. Otoliths first form in embryo and continue to grow throughout the life of an individual, with a double-banded increment composed of a calcium carbonate-rich region and a protein-rich region being deposited daily. In addition to this, they are considered to be metabolically inert, and do not undergo remodelling or resorption. Consequently, otoliths are employed in a variety of ways in fish ecology. Firstly, an individual fish’s age and growth rate can be estimated through counting increments and measuring their widths. Secondly, analysis of increment trace element:calcium ratios, such as by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), can allow for the reconstruction of environmental histories, aiding in the determination of natal origin, movement, habitat use, diet and the impacts of climate change. The utility of specific trace elements as indicators of environmental change, however, is unclear as there is considerable uncertainty as to whether a given trace element is interacting with the mineral or protein components of an increment. This uncertainty is a consequence of otolith research having been largely focussed upon either microstructure or inorganic chemistry, with very few studies on the protein-rich regions of the otolith. As a result, very little is understood about the biochemical mechanisms of biomineralization or trace element incorporation. This is important, as the mechanisms that govern otolith formation and growth underpin the assumptions made in traditional increment analyses. In this thesis, I initially undertook a systematic review of all the literature pertaining to otolith biochemistry, revealing the significant gaps that exist in otolith biochemistry as a discipline. Importantly, I determined that fewer than a score of otolith proteins had been identified – a stark contrast to the hundreds or thousands of proteins that have been identified in comparable biomineral systems such as enamel or bone. Working on black bream (Acanthopagrus butcheri), an extensively studied species endemic to southern Australia, I used size exclusion chromatography coupled with ICP-MS to determine the trace element:protein interactions in endolymph, the inner ear fluid that otoliths are submerged in, and the source of all of its constituents. In this study, I assayed 22 elements, and determined that 12 were solely present in a protein-bound form, 6 were present as free ions, and 4 were present in both forms. This allowed me to make recommendations as to their utility in environmental reconstructions. In my next study, I created a unique, multi-disciplinary workflow that combined transcriptomics with proteomics. In this study, I sequenced the transcriptome of the black bream inner ear and used this to identify proteins from the separated organic phase of otoliths and endolymph from wild caught adult black bream. This resulted in the discovery of hundreds of previously unknown proteins, providing new insights into the likely biochemical mechanisms involved in otolith formation and growth. In my final study, I tested the utility of trace element ratios in environmental reconstructions. Specifically, I compared the ability of different cluster analysis approaches to resolve spatial and temporal differences in the likely spawning and larval nursery habitats of juvenile black bream in the Gippsland Lakes, Australia. The results from my thesis have allowed me to make recommendations as to the utility of trace elements in environmental reconstructions and have revealed exciting new avenues of research that fuse ecology and biochemistry.
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ItemThe effects of chronic methamphetamine exposure during adolescence on brain and behaviour( 2018)Methamphetamine is a highly addictive psychostimulant that is used world-wide. The age of initial methamphetamine use typically occurs during adolescence, which is a particularly vulnerable period to the development of addiction. Therefore, this thesis aimed to elucidate the effects of methamphetamine during adolescence compared to adulthood on brain and behaviour. My first study examined the effects of either experimenter-injected binge exposure or self-administration of methamphetamine on subsequent fear related behaviours because the cycle of anxiety following methamphetamine use and withdrawal may be different in adolescence, which may contribute towards their methamphetamine use. In the binge model, adolescent and adult rats were injected with high and increasing doses of methamphetamine followed by fear conditioning. Extinction recall was impaired due to methamphetamine in adults but not adolescents. Methamphetamine self-administration did not differ between adults and adolescents, but it caused a deficit in the acquisition of conditioned fear in adults but not adolescents. In summary, prior methamphetamine exposure had effects on fear conditioning and extinction only in adults, but not in adolescents. My second study examined methamphetamine-cue extinction and cue-induced reinstatement following methamphetamine self-administration. While cue extinction reduced cue-induced reinstatement in adults and adolescents, adolescents showed higher cue-induced reinstatement compared to adults following 2 sessions of cue extinction. This chapter further showed that while adolescent and adult rats acquire methamphetamine self-administration similarly when the dose starts at 0.03 or 0.01 mg/kg/infusion, adolescents increase their methamphetamine intake when dose is increased from acquisition. This suggests that adolescents may be more vulnerable to escalate their methamphetamine intake if the dose is increased. My final study investigated the potential neuroadaptations induced by methamphetamine self-administration in adult and adolescent rats. Genome wide transcriptome analysis of the dorsal striatum identified 30 potential candidate genes with significant RNA expression changes due to methamphetamine. Based on ingenuity pathway analysis, 6 genes were followed up for quantitative real-time polymerase chain reaction validation. The most notable finding was that methamphetamine self-administration caused a decrease in solute-carrier family 18 member a1 (slc18a1) in adolescents but not in adults. The changes in the level of protein encoded by this gene, vesicular monoamine transporter 1 (VMAT1), were further validated by western blot. This chapter identifies factors that may explain adolescent vulnerability to addiction, such as their resistance to cue extinction and escalation of intake with dose increase. Age specific changes in gene expression following methamphetamine self-administration have been observed, which may explain the age-differences in methamphetamine-taking and seeking. Ultimately, these discoveries may provide novel ways to treat methamphetamine addiction depending on the age of onset of methamphetamine use.
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ItemMetal regulation of extra-neuronal tau( 2018)Metal dyshomeostasis is an important neurodegenerative event that can affect the structure and function of proteins, such as tau. Tau binds metals including copper, iron and zinc, and may undergo conformational changes that promote its aggregation, leading to neurodegeneration. Tau is mainly an axonal microtubule stabilising protein. However, tau is also located to dendrites where it mediates the transport of proteins to the postsynaptic terminal and may modulate the neuronal susceptibility to excitotoxicity. Furthermore, tau is present in bodily fluids, which can be used for detection of pathological biomarkers. The finding of tau in extracellular fluids and dendrites has directed its current research focus to the study of tau mechanisms of secretion and spreading. The aim of this project is to investigate the role of metals in the modulation of tau secretion, and to analyse if platelet tau and metal levels could serve as neurodegenerative blood biomarkers. To determine if metals could modulate tau secretion, primary cortical cell cultures from tau-knockout and wild-type mice were treated with copper, iron, zinc, glutamate and clioquinol, an 8-hydroxyquinoline derivative with ionophore activity. The results showed that copper, iron and clioquinol decreased tau secretion from cells whilst zinc and glutamate increased it. Copper and iron decreased APP secretion from cells whilst glutamate and clioquinol increased it. Additionally, tau-knockout cells showed decreased APP secretion compared to wild-type controls and metals did not alter their APP secretion. To further investigate if this occurs also in vivo, interstitial fluid (ISF) was collected using microdialysis on wild-type mice fed either with copper, iron or zinc. Zinc and iron increased tau ISF levels whilst copper decreased it. The analysis of baseline ISF tau levels collected over time from untreated animals suggested that tau could be involved in circadian rhythm. To evaluate if Alzheimer’s disease (AD) platelets present tau abnormalities and metal dyshomeostasis, platelet samples from 55 healthy and 45 AD subjects from the Australian Imaging, Biomarkers and Lifestyle flagship study of ageing (AIBL) were analysed by inductively coupled plasma-mass spectrometry (ICP-MS) and Western blotting. The results showed that copper is decreased in AD platelets. Additionally, healthy controls who are ApoEε4 carriers had the highest platelet copper content. Iron levels were decreased in AD platelets. Platelet zinc levels positively correlated with verbal fluency. ROC curves showed that copper and iron were poor biomarker candidates. There were no changes in total and GSK3β pS9 levels in AD platelets. To characterise platelet tau species, mouse brain and platelets from human, wild-type and tau-knockout mice were analysed by Western blotting and immunofluorescence. The results showed that tau immunoreactivity might be an antibody artefact. To further investigate this, tandem-MS was used to identify tau in a human platelet sample, using brain samples from wild-type and transgenic tau overexpressing mouse as positive controls. Tau was identified in mouse brain samples but not in human platelets. Additionally, a qRT-PCR was performed to platelets from humans, wild-type and tau-knockout mice to determine tau mRNA levels. The results showed that MAPT was expressed in wild-type and tau-knockout mice tissues but was not detected in either human or mouse platelets. The conclusions drawn from this thesis are that: (1) extracellular zinc may modulate tau secretion; (2) APP secretion may be tau dependent; (3) in AD platelets, there was no detectable level of tau and there was no change in GSK3β levels and activity; (4) platelets from AD patients exhibit metal dyshomeostasis, resulting in decreased copper and iron content. This change in platelet copper and iron concentrations alone do not provide sufficient sensitivity and specificity as AD biomarkers. Further studies are required to identify a panel of platelet biomarkers, together with changes in copper and iron that will provide high sensitivity and specificity as biomarkers for AD.
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ItemPotassium channel gene mutations in epileptic encephalopathy( 2018)The worldwide prevalence of epilepsy is between 2-3 % with many unmet clinical and health policy needs, especially in developing countries. Current estimates suggest that as many as 70 % of all epilepsy syndromes have a robust genetic etiology although the mechanisms of disease genesis are largely unknown. The improvement in genetic sequencing and testing has uncovered many genes associated with various forms of epilepsy, especially syndromes with an early onset. Many of these genes encode various ion channels, including the family of voltage-gated potassium channels, which are crucial in setting the afterhyperpolarization in neurons, preparing them to fire more action potentials. Mutations in these channels therefore bring about imbalance in the excitatory-inhibitory homeostasis in neural networks, leading to seizure development. Therefore the aim of this thesis was to characterize novel mutations of two different potassium channels encoded respectively by the KCNT1 and KCNC1 gene. The goal was to identify the biophysical changes brought about by KCNT1 and KCNC1 mutations, which were previously not reported. This thesis also looks at the effect of quinidine, which was previously reported as a possible therapeutic for KCNT1-related epilepsy, to determine the drug’s efficacy on these novel KCNT1 mutations. This thesis reports that the novel KCNT1 mutations identified from patients with autosomal dominant nocturnal frontal lobe epilepsy and epilepsy of infancy with migrating focal seizures produced potassium currents with large amplitudes and altered kinetics, similar to previously reported KCNT1 mutations in the literature; however, there was no clear genotype-phenotype pattern identified. Novel KCNT1 mutations in myoclonic atonic epilepsy and Lennox-Gastuat Syndrome, where changes in the KCNT1 gene were previously unreported, produced similar biophysics to WT, therefore indicating that KCNT1 may not be the underlying cause of epilepsy in these patients. The inclusion of single nucleotide polymorphisms in these experiments acted as controls as well as emphasized that not all genetic alterations are detrimental. The application of quinidine on some of the variants showed varied effects with some mutations having reduced and some increased currents upon its application, indicating that the drug may not be a magic-bullet treatment for all KCNT1-related epilepsies. This thesis also looked at characterizing novel KCNC1 mutations identified in patients with epileptic encephalopathy or intellectual disability without seizures. These de novo variants were compared against the recurrent p.Arg320His mutant, which was previously identified in patients suffering from progressive myoclonus epilepsy. The results obtained show varied levels of loss of function for the analysed mutants with some of them also showing changes in voltage dependence of activation and a dominant-negative effect. Therefore the results obtained in this thesis, provide a basic insight into the changes caused by newly identified mutations in two voltage-gated potassium channel genes. The data act as a framework, which can assist in the development in more complex experimental models to further understand the biophysical effects of the mutation as well as drug-protein interactions. Nonetheless, this work also emphasizes the need for in vitro experiments as a way of breaking down complex disorders such as epilepsy.
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ItemThe role of neuroligin 3 in cognitive function( 2018)Neuroligins (NLGNs) are members of trans-synaptic protein complexes that mediate synaptic transmission and plasticity, both of which are critical for information processing and cognition. Genetic risk factors for a range of neurodevelopmental disorders disproportionately converge upon synaptic genes, including NLGNs. Although mutations in NLGN3 are linked to ASD, a condition with characteristic cognitive impairments, little is known about the impact of a loss of function of NLGN3 on cognitive processing. This thesis describes the impact of a loss of function of Nlgn3 on a range of distinct cognitive functions in mice, using rodent touchscreen technology. In Chapter 2, we assessed Nlgn3 KO mice on a battery of cognitive tests to explore the involvement of Nlgn3 in operant learning, associative learning, behavioural flexibility, response inhibition and motivation. We found that loss of function of Nlgn3 did not impact operant learning, discrimination learning, extinction learning or appetitive motivation, but had striking impacts on behavioural flexibility indexed by perseverative incorrect responding as well as performance on a reversal learning task. In Chapter 3 we extended the characterisation of Nlgn3 KO mice to probe working memory, pattern separation, signal detection and sustained attention. We found that Nlgn3 KO mice displayed normal pattern separation and a subtle but significant impairment in working memory, suggestive of a shift in the balance between stability and flexibility. Loss of Nlgn3 additionally resulted in robust improvements in measures of sustained attention and signal detection, implicating Nlgn3 in processes underlying allocation of attention. However, performance of Nlgn3 KO mice was also more severely impacted by increasing demands on perceptual or attentional processing, suggesting a reduced ability to scale up the allocation of processing resources under challenging conditions. In Chapter 4 we examined relational memory and flexibility by adapting a novel version of the touchscreen transitive inference task. After validating that WT mice were able to perform our adapted version of the touchscreen transitive inference test, we assessed both Nlgn3 KO and Nlgn3 R451C mice to compare two different models of Nlgn3 dysfunction on relational memory. We show that both Nlgn3 KO and Nlgn3 R451C mice display intact relational memory. However, both Nlgn3 mutant mice exhibited elevated behavioural flexibility, consistent with our results from Chapter 2. Across all chapters, our results indicate Nlgn3 KO mice exhibit longer response times than WT mice across tests that require discrimination of complex stimuli, strongly implicating Nlgn3 in processes underlying perceptual processing. Collectively, the findings from this thesis show that Nlgn3 is involved in aspects of behavioural flexibility, attention and perceptual processing. These studies contribute to our understanding of the function of Nlgn3, and more broadly the impact of synaptic gene dysfunction in aspects of cognition relevant to neurodevelopmental disorders.
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ItemUndetected preclinical neurodegenerative disease in models of normal cognitive aging( 2018)With population aging, understanding cognitive changes that occur in late life is vital to support these increasing numbers of older adults to maintain their wellbeing and independence. Furthermore, accurate estimates of age-related cognitive change will enable the differentiation of early stage neurodegenerative disease from normal aging. Current cognitive aging models describe a pattern of progressive decline in memory,executive function, and processing speed abilities, and retention of experience-based knowledge, with increasing age. However, given that many older adults show signs of neurodegenerative disease, despite not meeting clinical criteria for dementia, it is possible that cognitive aging studies may have over-estimated the nature and magnitude of age-related cognitive decline. The aim of this thesis was to determine the extent to which undetected preclinical neurodegenerative disease could influence models of cognitive aging. Age-related change in cognition was examined in cognitively normal healthy older adults who underwent repeated clinical and neuropsychological assessments, as well as biomarker assessment for neurodegenerative disease. The influence of progression to mild cognitive impairment (MCI) and dementia was also considered. Results indicated that estimates of age-related cognitive decline were inflated by undetected disease. This was also found when the data were reconceptualised as intelligence factors and was confirmed across two cohorts and utilizing a range of analytic methods. Notably, in the absence of disease, increasing age was associated with stability of performance in episodic and working memory, and an attenuated rate of decline in some processing speed and executive functions. Together these results indicate that current expectations about cognitive loss in aging are biased by unrecognized neurodegenerative disease.