Late-onset Alzheimer’s disease (LOAD) is the most common form of dementia, with an incidence of up to 50% in western populations over the age of 85 and a high heritability (up to 80%). The identification of risk factors for the development of LOAD is imperative for improving our understanding of this disease and for identifying therapeutic targets for treatment or prevention. Currently, the major known risk factors for the development of LOAD are age and the ApoE ε4 genotype. Previous studies have implicated plasma levels of the amyloid beta (Aß) peptide as a LOAD-associated quantitative trait and identification of loci influencing this trait could provide new insights into LOAD. In this thesis, plasma levels of the Aß peptides Aß40 and Aß42 have been measured in two isolated populations and genome-wide linkage and association analyses were performed. The genome-wide association analyses identified a number of promising quantitative trait loci; highlighting both novel and previously reported LOAD genes for further study, whilst also providing an excellent resource for genetic convergence studies with other LOAD related traits. Several studies have reported an association between levels of oxidative stress and levels of Aß such that increasing levels of Aß appear to increase markers for oxidative stress and vice versa. The role of oxidative stress in LOAD and aging was therefore also investigated through analysis of mitochondrial mutational burden and DNA damage respectively, using DNA isolated from both blood and the brain and by carrying out a candidate gene association study of loci involved in mitochondrial function in LOAD cases and controls. Approaches to the investigation of mitochondrial genetics for the study of LOAD are comprehensively reviewed, adapted and tested and the results indicate a need for additional research in this aspect of the disease. This thesis therefore presents a focus on two aspects of genetic research into LOAD, a complex disease with multiple environmental and genetic influences which aims to advance our understanding of the disease and bring us closer to treatment and prevention strategies.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder associated with aging. AD is by far the best understood and most studied neurodegenerative disease. Substantial advances have been made over the last decade, however it is debatable how much closer we are to a clinically useful therapy. A long standing goal in the AD field has been to improve the accuracy of early detection, with the assumption that the ability to intervene earlier in the disease process will lead to a better clinical outcome. Major facets of this effort have been the continued development and improvement of AD biomarkers, with a strong focus on developing imaging modalities. AD is accompanied by two pathological hallmarks in the brain: extracellular neuritic plaques composed of the beta-amyloid peptide (Aβ) and intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein. Evidence of Aβ as the driving force behind the progression of AD (the amyloid cascade hypothesis) was first published by Hardy & Higgins in 1992, and this peptide has been the focus of therapeutic and diagnostic testing for decades. Significant technological advances in recent years now allow imaging of amyloid pathology in vivo. These methods evaluate Aβ burden in a living person, and could potentially serve as both a biomarker, and as a diagnostic tool to detect disease. Pittsburgh Compound B (PiB) is currently the best studied of these imaging agents, however, our current knowledge of the quantitative relationship between PiB binding and amyloid pathology in the brain is limited. A better understanding of how these variables relate to one another is essential for the continued development of reliable diagnostic biomarkers for AD. We analyzed increasingly insoluble pools of Aβ to quantify their relative contributions to the overall Aβ burden, and to determine if any of these measures could be used to predict disease status. We found that the amount of PiB binding in a cortical region of the brain could distinguish cases of mild cognitive impairment (MCI) when corrected to the amount of PiB binding in the cerebellum. As the Aβ peptide ages, the amino acid aspartate may spontaneously convert to an isoaspartate residue through a succinimide intermediary. The presence of iso-Asp Aβ has been used to indicate the presence of aged plaques in AD and Down syndrome cases. We sought to investigate the potential relationship between levels of ‘aged’ Aβ in the plasma as indicated by iso-Asp Aβ and disease state, as a potential biomarker for the presence of AD pathology. We found that AD cases had lower levels of all forms of Aβ in plasma when standardized to the group average, and that plasma levels of Aβ and iso-Asp Aβ were reversed between disease groups. A follow up study is required, however, these initial data are a promising step towards utilizing aged iso-Asp Aβ plasma levels as a potential biomarker to indicate disease state.

Alzheimer’s disease (AD) is a very complex disorder and the most common form of dementia. The two pathological hallmarks of AD are extracellular amyloid-β (Aβ) plaques in cerebral cortex, and intraneuronal neurofibrillary tangles. In the early stages of the disease it can be difficult to accurately diagnose AD, as it is difficult to distinguish from normal signs of aging. There is thus a need for sensitive non-invasive tools, able to detect pathophysiological biomarker changes. One such approach is molecular imaging of Aβ plaque load in brain, using PET (positron emission tomography) ligands. We have developed and characterized two novel Aβ plaque neuroimaging PET ligands, AZD2184 and AZD4694. The 2-pyridylbenzothiazole derivate AZD2184, is a 11C-labeled PET ligand with a higher signal-to-background ratio compared to the widely used PET ligand PIB, a 11C-labeled phenylbenzothiazole based tool. This makes it possible to detect smaller changes in Aβ plaque deposition load, and therefore theoretically, also earlier diagnosis. A drawback with 11C-labeled PET ligands is the relatively short half-life. To meet the need for PET ligands with a longer half-life, we developed the pyridylbenzofuran derivate [18F]AZD4694. Although development of fluorinated radioligands is challenging due to the lipophilic nature of aromatic fluorine, we successfully developed a 18F-labeled PET ligand with a signal-to-background ratio matching PIB, the most widely used 11C-labeled PET ligand in clinical use. 3H-labeled derivates of AZD2184, AZD4694, and PIB, showed lower binding specificity towards Aβ plaques containing ApoE. The ApoE genotype per se did not significantly affect ligand binding, instead, the amount of ApoE incorporated to the Aβ plaques appears to be of importance for the binding characteristics of these amyloid PET ligands. Beta-secretase 1 (BACE1) mediates the first step in the processing of amyloid precursor protein (APP) to Aβ peptides, making BACE1 inhibition an attractive therapeutic target in AD. We developed and characterized three novel BACE1 inhibitors, AZD3839, AZ-4217, and AZD3293. AZD3839 and AZ-4217 contains an amidine group which interacts with the catalytic aspartases Asp-32 and Asp-228 of BACE1, effectively inhibiting the enzyme. All three compounds are potent and selective inhibitors of human BACE1, with in vitro potency demonstrated in several cellular models, including primary cortical neurons. All three compound exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aβ levels in several species, and two of the compounds (AZD3839 and AZD3293) were progressed into clinical trials. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Submitted.</p>

Patients with Alzheimer’s Disease (AD) demonstrate a sensitive and specific short-term memory impairment for visual bindings (e.g. the combination of shapes and colours) that is absent in healthy ageing (Parra et al., 2009) and other dementias (Della Sala et al., 2012). This impairment is also seen in cases of asymptomatic, familial AD (Parra et al., 2010). The visual short term memory binding (VSTMB) impairment in AD has clear clinical and neuropsychological implications which are investigated in this thesis. Firstly, the utility of the VSTMB paradigm was contrasted with the Free and Cued Selective Reminding Task with Immediate Recall (FCSRT-IR) – which has recently been posited as a useful diagnostic marker of AD pathology (Dubois et al., 2014). The results indicated that the former is not affected by age, where the latter is, suggesting that the VSTMB paradigm provides a suitable baseline to measure cognitive decline associated with AD. The development of a parallel version of the FCSRT-IR is also reported. Secondly, a 24-week longitudinal study of patients receiving treatment for AD (donepezil hydrochloride) revealed that patients who respond to this treatment on cognitive scales also experience change in VSTMB performance. These responders, however, did not significantly improve on the FCSRT-IR during the study. This suggests that anticholinergic treatment may have an effect on VSTMB performance. Additionally, a meta-analysis that investigates the effect of a study’s funding on donepezil RCT outcome showed that industry-funded studies report larger changes in cognition than independent studies. Lastly, an auditory binding experimental paradigm was developed, with a view to reveal a non-visual binding impairment in AD, investigating whether the binding impairment reflects a general or modality-specific memory impairment. The overall conclusions of this thesis confirm that the VSTMB impairment has significant promise as an index of AD. The auditory binding paradigm, by contrast, shares conceptual similarity with the VSTMB paradigm, but may have restricted clinical use within the AD patient population.

Introduction The present study explored the nature of the semantic deterioration normally displayed in the course of Alzheimer’s disease (AD). The aim was to disentangle the extent to which semantic memory problems in patients with AD are best characterized as loss of semantic knowledge rather than difficulties in accessing semantic knowledge. Method A longitudinal approach was applied. The same semantic tests as well as same items were used across three test occasions a year apart. Twelve Alzheimer patients and 20 matched control subjects, out of a total of 25 cases in each group, remained at the final test occasion. Results and Conclusions Alzheimer patients were impaired in all the semantic tasks as compared to the matched comparison group. A progressing deterioration was evident during the study period. Our findings suggest that semantic impairment is mainly due to loss of information rather than problems in accessing semantic information.

Alzheimer’s is a widespread neurodegenerative disease, growing larger and larger in the world. Once developed, the disease has no cure. To this day, there is only mitigating drugs. To be able to start this treatment rapidly, a method to distinguish healthy individuals from prospective Alzheimer’s diseased needs to be developed. Cerebrospinal fluid is thought to contribute to the development of such method, through its close substitution of fluids, molecules and proteins from the brain. It may provide a progressive marker of the disease, a substance differently expressed in the healthy and diseased; a biomarker. The aim of the present study was to develop and evaluate a stable method for degradation and analysis of peptides and proteins in the cerebrospinal fluid using mass spectrometry techniques, such as selective reaction monitoring. Mass spectrometry is often used after a first dimension separation with liquid chromatography. Three degradation methods were evaluated, resulting in a protocol with the detergent DOC being the most beneficial. Tryptic peptides occurred in a concentration of 10 % w/v due to the SDS-PAGE gels and database searches concomitantly. The elution pattern from the liquid chromatography enables a narrow selection in the sensitivity for each peptide. Chromatographic preferences such as column, hydrophobicity and time span was determined, and unwanted peptides filtered away. A specific protein, the amyloid precursor protein APP, is thought to play a significant part in the development of Alzheimer’s disease. The protein is located in the neurons, cleaved and processed to produce the neurodegenerating plaques found in the brain at the diseased. Three isoforms are found in the neurons, APP695, APP751 and APP770. When cleaved, a shorter soluble tryptic peptide is generated from all APP isoforms. This was the target for the current study, as a potential progressive biomarker. The method developed was able to separate and distinguish the soluble APP751 isoform, but not the APP695 or APP 770 isoforms, most probably due to glycosylations of the two resembling isoforms.

Dementia is characterized as a progressive loss of brain function that results in the deterioration of many cognitive and physical abilities. Alzheimer’s disease (AD) is the most common form of dementia, causing steady declines in memory, functional abilities, and mental functioning. With a projected increase of degenerative illnesses, such as AD, family caregiving for individuals with the disease is also steadily increasing. Caring for an individual with AD has been characterized as a “career,” and within this career are a number of key transitions, including the onset of caregiving. Preexisting caregiving research reveals a number of negative consequences for AD family caregivers, including depression, overload, and physical health complications. The purpose of this study was to examine how different patterns of caregiving onset (gradual and abrupt) and role occupancy (how many roles the caregiver is holding) impact mental health and physical health outcomes for AD caregivers. This study also explored how cognitive decline and behavioral problems found within the care-recipient have the potential to moderate these relationships. Cross-sectional, quantitative data from one hundred participants completing self-administered surveys was used in this study. A series of one-way ANOVAS and multiple regression analyses were conducted to address the study’s aims. Results indicated that care-recipient cognition and behavioral problems moderated the relationship between caregiving onset and mental health outcomes, including depression, role overload, and role captivity for caregivers who experienced a more abrupt entry into the caregiving role. Results suggest the importance of considering moderating factors within the caregiving career, as well as different caregiving onset transitions. Clinical implications of the findings are discussed, as well as directions for future research, including prospective caregiving research.

Human brain structure can be measured across the lifecourse (“in vivo”) with magnetic resonance imaging (MRI). MRI data are often used to create “atlases” and statistical models of brain structure across the lifecourse. These methods may define how brain structure changes through life and support diagnoses of increasingly common, yet still fatal, age-related neurodegenerative diseases. As diseases such as Alzheimer’s (AD) cast an ever growing shadow over our ageing population, it is vitally important to robustly define changes which are normal for age and those which are pathological. This work therefore assessed existing MR brain image data, atlases, and statistical models. These assessments led me to propose novel methods for accurately defining the distributions and boundaries of normal ageing and pathological brain structure. A systematic review found that there were fewer than 100 appropriately tested normal subjects aged ≥60 years openly available worldwide. These subjects did not have the range of MRI sequences required to effectively characterise the features of brain ageing. The majority of brain image atlases identified in this review were found to contain data from few or no subjects aged ≥60 years and were in a limited range of MRI sequences. All of these atlases were created with parametric (mean-based) statistics that require the assumptions of equal variance and Gaussian distributions. When these assumptions are not met, mean-based atlases and models may not well represent the distributions and boundaries of brain structure. I tested these assumptions and found that they were not met in whole brain, subregional, and voxel-based models of ~580 subjects from across the lifecourse (0- 90 years). I then implemented novel whole brain, subregional, and voxel-based statistics, e.g. percentile rank atlases and nonparametric effect size estimates. The equivalent parametric statistics led to errors in classification and inflated effects by up to 45% in normal ageing-AD comparisons. I conclude that more MR brain image data, age appropriate atlases, and nonparametric statistical models are needed to define the true limits of normal brain structure. Accurate definition of these limits will ultimately improve diagnoses, treatment, and outcome of neurodegenerative disease.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder and is the main cause of dementia in the elderly population. AD is pathologically characterized by the accumulation of amyloid plaques and neurofibrillary tangles that results in neurodegeneration and loss of memory function. However, diagnosis of AD and characterization of biological mechanisms that lead to pathology and modulate risk for disease has proven to be extremely difficult. Cerebrospinal fluid (CSF) contains critical biomarkers for AD such as levels of amyloid beta (Aβ) phosphorylated-tau (p-tau), total-tau (t-tau), and neurofilament light chain (NfL). The CSF levels of these biomarkers are useful in determining AD status in a patient, but data collection can be time consuming, technically difficult, and expensive. While still subject to the limitations of obtaining CSF, cell free single stranded DNA (cfssDNA) is much cheaper and more reliably measured than these biomarkers. We investigated cfssDNA as a biomarker for AD status. We observed an association between low levels of concentration isolated from CSF as a potential biomarker for diagnosis of AD. Inflammation is a vital process in the immune system. Acute inflammation plays an essential role in the normal response to tissue injury. This inflammatory response initiates a cascade of cellular activation signals in innate immune cells resulting in increased production of proinflammatory cytokines and chemokines. These chemokines are essential to the recruitment and activation of other cells in the innate and adaptive immune system. Deviations from the normal production of these chemokines can result in disease status. Recently published work has identified genetic variants that show strong associations with AD-related chemokine levels in CSF and plasma. We attempted to characterize the biological mechanisms that underlie the reported associations between the ACKR2-V41A variant and CCL2 levels and the CCRL2-V180M variant and CCL4 levels. Our data demonstrate that the ACKR2-V41A receptor has a lower CCL2 binding affinity, scavenging efficiency, and receptor upregulation compared to ACKR2-WT. For CCRL2-V180M our data demonstrate higher binding affinity with chemerin and CCL19 than CCRL2-WT. Our data also show that while CCRL2-V180M and CCRL2-WT do not directly bind with CCL4, interactions between CCRL2-V180M and CCL19 alter the secretion of CCL4 from leukocytes. These findings provide evidence for a novel biomarker for AD diagnosis, mechanistic insights into the functional impact of common genetic variants on chemokine levels, and highlight a potential role of atypical chemokines in altering the risk for AD.

Alzheimer’s disease (AD) patients could benefit from a more effective treatment than the current FDA-approved options. Because amyloid-beta (Aβ) is thought to play a central role in AD pathogenesis, many experimental drugs attempt to reduce Aβ-induced pathology. Preventing amyloid accumulation may be a more effective strategy than clearing Aβ plaques after they form. If preventing Aβ accumulation can treat or prevent AD, then understanding Aβ primary nucleation may aid rational drug design. This study examines Aβ residue interaction networks and reports network and structural observations that may provide insight into primary nucleation. While many studies identify structural features of Aβ that promote aggregation, this study reports features that may resist primary nucleation by examining Aβ42 studies in more and less polar solvents. In Aβ42 in a less polar solvent (PDB ID: 1IYT), Val24 and Ile31 have higher betweenness and residue centrality values. This may be due to a predicted interaction between Val24 and Ile31. Residues in the central hydrophobic cluster (CHC) of Aβ40 and Aβ42 had significantly higher betweenness values compared to the average betweenness of the structures, highlighting the CHC’s reported role in oligomerization. The predicted interaction between Val24 and Ile31 may reduce the likelihood of primary nucleation of Aβ.