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Originally conceived of as a disease associated with old age, Alzheimer's disease (AD) is now considered to have its earliest origins in midlife. There is considerable interest in understanding more about modifiable risk factors throughout the life span, which may delay or even prevent symptom onset. Glucocorticoids (cortisol and cortisone) are our stress hormones and may be important modifiable risk factors for AD. Cortisol is physiologically active, while cortisone is inactive, recycled to cortisol by the action of the 11β-hydroxysteroid dehydrogenases. Previous research studies have focussed on cortisol and have reported associations between higher levels of cortisol and faster cognitive decline, higher AD pathology and a faster transition from the preclinical to the dementia stage of AD. These studies have typically analysed cortisol only using immunoassays, rather than the gold-standard technique of liquid chromatography tandem mass spectrometry (LC-MS/MS). There is currently limited evidence investigating associations between glucocorticoids in the preclinical stage of AD, with no studies focusing on cortisone. Cortisol abnormalities have also been reported in a number of conditions that are known risk factors for future AD, including diabetes, obesity, depression, anxiety and trauma. Studies have not yet explored whether cortisol is a mediator between these risk factors and AD. The aims of this thesis are [1] to establish a method for the analysis of a multi-steroid panel in saliva samples collected from older adults [2] to explore associations between diabetes and cognition investigating glucocorticoids as a mediator, and [3] investigate cortisone as a modifiable risk factor for AD. This thesis uses data and samples from two established cohort studies; the European Prevention of Alzheimer's Dementia (EPAD) Longitudinal Cohort Study (LCS) (n=2094, aged 50+ years) and EPAD Scotland (n=15, aged 50+ years). Steroids in saliva samples (200 µL) from 125 participants enrolled in the EPAD LCS were analysed using an LC-MS/MS method developed for this thesis. The LC-MS/MS method was determined to be feasible over physiological ranges and data analysis confirmed that cortisone is the more abundant and suitable glucocorticoid than cortisol to use when working with saliva samples. These results highlight the need for chromatographic separation of glucocorticoids prior to detection. Linear regression analysis identified a statistically significant association between diabetes and performance on the Four Mountains Test (4MT; a task of allocentric processing) in the EPAD LCS (fully adjusted model β: -0.57 p=0.04, 95% CI: -1.11, -0.03). This association was not explained by salivary cortisone in the EPAD LCS. The results suggest that while diabetes is associated with the earliest stages of cognitive impairment, this is not mediated via glucocorticoids, suggesting a different path to pathology accumulation. The salivary cortisone slope was associated with continuous CSF Aβ42 levels in partially and fully adjusted models in a subset of EPAD LCS participants (n=125). When the dataset was split around the median into higher and lower cortisone, there was a significant association between log-transformed cortisone AUC and CSF Aβ42 negativity in all models in the higher cortisone group. Intra-individual variability of either cortisone AUC or slope were not significantly associated with Aβ42 or pTau181. These findings suggest that having more inactive glucocorticoid in the saliva, but not variability of this metric, is associated with better brain health, which may provide an opportunity for intervention. This PhD provides evidence that [1] using LC-MS/MS it is feasible to detect multiple steroids in 200 µL saliva samples from older adults; [2] that there is a significant association between diabetes and risk for dementia in midlife and poorer cognitive performance in later life in two at-risk for dementia cohorts; [3] that the association between diabetes and cognition was not mediated by glucocorticoids; [4] that there is a significant association between salivary cortisone and CSF Aβ42; and that [5] there was no association between intra-individual variability of cortisone over four days of sampling and brain health. The final chapter discusses future work, limitations possible mechanisms and clinical implications.

Many neurodegenerative diseases are characterized by the accumulation of misfolded proteins that often share common morphological and biochemical features, and can similarly co-localize with several other proteins, including various chaperone proteins. Chaperone proteins, like heat shock protein 27 (HSP27), heme oxygenase 1 (HO-1) and clusterin, have been implicated as potent modulators of misfolded proteins, thus may play important roles in the pathogenesis of neurodegenerative diseases. The present study aims to investigate their roles in the pathogenesis of Frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), Parkinson's disease (PD), and Motor neuron disease (MND) by determining their distribution and amount via immunohistochemical staining and western blotting in diseased and control subjects.There were distinct patterns of HSP27 and clusterin immunostaining in different brain regions. For HSP27, patients with AD and FTLD were in general more severely affected than were patients with MND and control subjects. For clusterin, patients with AD and FTLD were more severely affected than control subjects where neurons and glial cells were concerned, while patients with AD and control subjects were more severely affected than those with FTLD where diffuse and cored plaques were concerned. However, there were no obvious differences in the pattern of HO-1 immunostaining in various brain regions in patients with AD or FTLD relative to control subjects. Moreover, there was no association between HSP27, HO-1 and clusterin with disease or histological type, and the ‘classic’ neuropathological changes in FTLD, AD and MND were not immunoreactive to any of these proteins. There were significant correlations between the degrees of HO-1 and clusterin immunostaining in many brain areas for both AD and FTLD cases, and for all cases overall, but none between HSP27 and clusterin or HSP27 and HO-1. Present results suggest an involvement with ongoing cellular stress, misfolded or unfolded protein accumulation or the deficits/failure of other relevant protein quality control systems, in the pathogenesis of these neurodegenerative diseases. Present work may therefore have implications for the further development of ideas concerning the cause or treatment of neurodegenerative diseases where there is aberrant accumulation of misfolded, aggregated protein, and perhaps for conformational diseases in general. However, there are still many issues remain to be elucidated. Further research aimed at understanding the function and mechanisms of the chaperone system, and other protein quality control mechanisms, in the pathogenesis of neurodegenerative diseases is still needed.

In this dissertation, two mass spectrometry techniques, covered in Chapter 1, have been employed for qualitatively and quantitatively characterizing protein-derived carbohydrate moieties, including N- and O-glycopeptides as well as free glycans derived from biological specimens. In Chapter 2, an RP-LC-MS/MS system functionalized with multiple fragmentation performances, specifically CID and HCD, is utilized to survey all probable N- and O-glycosites along the backbone of a coagulation factor, human plasma von Willebrand Factor, with full characterization of covalently linked N- and O-glycoforms. N-glycosite occupancy varied along the protein backbone chain. 181-characterized glycoforms are specified into either N- or O-glycosites. Noteworthy, two previously unreported N-glycosites within domain D’(TIL’-E’) are occupied with N-glycoforms. In Chapter 3, MALDI-TOF technique is utilized to explore aberrant N-glycosylation in the frontal cortex (FC) region extracted from Alzheimer Disease (AD) patients’ brains in comparison with age-matched individuals. AD FC N-glycome exhibits a significant decrease in galactosylation and fucosylation as well as sialylation compared to normal FC. Eight N-glycoforms, detected across all cases, are significantly differentiated between the examined cohorts. Also, high mannose, hybrid, complex, and truncated N-glycans are considerably changed in disease tissues. In Chapter 4, altered N-glycosylation of soluble serum glycoproteins derived from colon cancer (CC) patients’ blood sera is characterized by MALDI-TOF. N-glycome of CC glycoproteins shows an increase in aglactosylation, fucosylation, and sialylation, especially sialylated core-fucosylated species. The knowledge of this work (Chapter 5) might improve our perception of disease pathogenesis or enhance the efficacy of current therapy treatments.

Translocator protein (TSPO) is thought to be fundamental for mitochondrial transmembrane cholesterol transport, other mitochondrial functions and brain inflammation. It is upregulated in glial cells during inflammation, for example in Frontotemporal Lobar Degeneration (FTLD) and Alzheimer’s disease (AD). High-affinity TSPO radioligands are utilised in inflammation imaging. However, this is hampered due to the A147T polymorphism which modulates ligand binding. The polymorphism also has been linked to increased risk of certain neuropsychiatric disorders, possibly as it reduces TSPO protein stability. TSPO was originally discovered as a benzodiazepine receptor: there is now controversy concerning TSPO hypothesised functions as evidence for these functions may have resulted from drug or ligand off-target effects. Moreover, recent knockout studies have challenged these roles. In light of these controversies, I investigated TSPO protein function in TSPO knockout transgenic mouse models of FTLD and AD. Firstly, I investigated TSPO’s role in reactive gliosis or neuroinflammation in the presence of tau pathology. TSPO knockout mice were crossed with TAU58/2 mice (tau P301S mouse model of FTLD). TSPO deletion ameliorated tau-induced pathological and behavioural deficits in TAU58/2 mice. Mechanistically, TSPO knockout reduced microglia activation, resulting in less tau deposition. Secondly, I investigated TSPO’s role in neuroinflammation in the presence of amyloid beta (Aβ) pathology. TSPO knockout mice were crossed with both APP23 mice and APP NL-G-F mice (two different AD transgenic mouse models). TSPO depletion delayed Aβ-related pathology and reduced microglia activation in APP23 mice. Intriguingly, depleting TSPO in APP NL-G-F mice had opposing effects. Taken together, TSPO knockout confers protection against tau and Aβ pathogenesis through suppression of microglia activation. However, this effect is age- and model-dependent. Finally, using immunoprecipitation coupled to mass-spectrometry, I identified 30 TSPO protein-protein interactions (PPIs), yet only 23 A147T PPIs, indicating that A147T polymorphism of the TSPO might confer loss of function. This thesis reveals TSPO as a positive regulator of microglia activation in mouse models of FTLD and AD. Strategies targeting microglia to suppress their activation, possibly through the use of TSPO-specific inhibitors, might provide alternative disease-modifying targets for FTLD and AD.

The risk of suffering from Alzheimer’s disease (AD) is higher in individuals from AD-affected mothers. The purpose of this investigation was to study whether maternal transmission might produce AD-related alterations in progenies of mice that do not have any genotypic alteration. We used cognitively-intact mothers harbouring in heterozygosity the transgene for overexpressing the Swedish double mutant version of the human amyloid precursor protein (hAβPPswe). The phenotype of the offspring with or without the transgene resulting from crossing young Tg2576 females with wild-type males were compared with those of the offspring resulting from crossing wild-type females with Tg2576 males. The hAβPPswe-bearing offspring from Tg2576 mothers showed an aggravated AD-like phenotype. Remarkably, cognitive, immunohistochemical and some biochemical features displayed by Tg2576 heterozygous mice were also found in wild-type animals generated from Tg2576 females. This suggests the existence of a maternal imprinting in the wild-type offspring that confers a greater facility to launch an AD-like neurodegenerative cascade. Such progeny, lacking any mutant amyloid precursor protein, constitutes a novel model to study maternal transmission of AD and, even more important, to discover early risk markers that predispose to the development of AD.

Alzheimer’s disease has been described long ago, yet the illness is yet to be fully understood. Though it is true that research and technological advancements have brought us closer to understanding the disease, a truly effective pharmacological cure has not been discovered. With no permanent cure to rely on, AD patients and their caregivers still go through profound struggles in navigating through life with the disease. In this thesis, current literature on the non-pharmacological interventions is presented and discusses the various options that can provide the greatest relief and reap the most health benefits for patients. In total, four different non-drug treatments come into discussion - exercise, music, diet and cognitive interventions. In terms of exercise, research suggests that anaerobic work may be more beneficial than aerobic exercises in preventing the development or progression of mild cognitive dementia and Alzheimer’s disease. This is mostly due to the fact that anaerobic exercises can shift APP processing away from the non-amyloidogenic pathway and increase BDNF levels to offer improved neural protection. Music therapy intervention is evaluated next. This unique treatment is highly valued due to its beneficial effects on AD patients’ emotional well-being. Music therapy can take the forms of singing in groups or as an individual, and it can also incorporate dancing. Not only does music promote neuroplasticity and neurogenesis, but it also alleviates mood, boosts confidence and strengthens will. Diet is another significant component that can have an incredible impact on the AD patients’ wellbeing. Research reveals that diets high in saturated fatty acid should be avoided. On the other hand, diets mirroring the Mediterranean diet, including polyunsaturated fatty acids along with high amounts of vitamin C and folic acid should be readily consumed. Moreover, spices and herbs such as capsaicin should be used in a limited manner to decrease risk for AD. Finally, cognitive therapy is still a popular method for treating mild cognitive impairment and AD. Though cognitive improvement appears to be more modest, some psychostimulation programs combined with pharmacological treatments can play an influential role in achieving cognitive stability. Further research is needed in upgrading the current non-pharmacological interventions with an emphasis on the four treatments. These are available at an affordable cost and can be easily incorporated into the lifestyles of Alzheimer’s patients.

Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting over five million patients worldwide. Like Alzheimer’s disease (AD), it mostly affects the elderly and causes considerable disability and suffering. Unfortunately, the molecular mechanism of PD is still poorly understood, and there are no drugs available to treat the disease. Our overall aim was to identify natural products to probe PD by phenotypic assay using human olfactory neurosphere-derived (hONS) cells from PD patients. The research presented in this thesis exemplifies the importance of natural products as chemical probes for further investigation of PD as well as lead compounds for future PD-drug development. The thesis begins with an introduction of PD and the chemotherapeutics for PD. It also covers a review on the natural origin anti-PD compounds and the analysis of their physicochemical properties using Lipinski’s rule of five. As part of a research program aiming to identify anti-PD chemical probes, a high throughput screening assay was developed to screen 4224 fractions. Twenty fractions were confirmed to display neuroprotective effects of the PD cells against rotenone. Seven prioritized fractions, representing one Australian marine sponge Jaspis splendens (subject 1) and two Australian terrestrial plants Gloriosa superba (subject 2) and Alangium villosum (subject 3), were selected for large scale extraction and isolation. The results were presented in Chapter 2 to 5.

Previous studies from our laboratory and others show a significant increase in levels of both nuclear and mitochondrial DNA and RNA oxidation in vulnerable brain regions in the progression of Alzheimer’s disease (AD). Although total DNA oxidation is increased in AD it remains unclear whether oxidative damage is widespread throughout the genome or is concentrated to specific genes. To test the hypothesis that specific genes are more highly oxidized in the progression of AD, we propose to quantify the percent oxidative damage in genes coding for proteins shown to be altered in the progression of AD using quantitative/real-time polymerase chain reaction (qPCR/ RT-PCR). To further test the hypothesis that diminished DNA repair capacity in the progression of AD contributes to increased DNA oxidation we will use custom PCR arrays and qPCR, Western blot analysis and activity assays to quantify changes in enzymes involved in base excision repair (BER). In order to carry out these studies tissue specimens from superior and middle temporal gyri (SMTG) and inferior parietal lobe (IP), as well as, a non-vulnerable region, the cerebellum (CER) will be analyzed from normal control (NC) subjects and subjects throughout the progression of AD including those with preclinical AD (PCAD), mild cognitive impairment (MCI), and late stage AD (LAD). We will also analyze specimens from diseased control subjects (DC; Frontotemporal dementia (FTD) and dementia with Lewy bodies (DLB)) to determine if the changes we observe in AD are specific.

Alzheimer's   disease   (AD)   is   a   prevalent   degenerative disease that is both idiopathic and highly debilitating, calling for early detection and intervention before extensive irreversible brain damage occurs. Here, a novel functional complexity measure based on information theory was applied to mark a disease-related reduction in stochastic low frequency (0.01-0.08 Hz) BOLD signal fluctuations at rest. As AD progresses, BOLD signal complexity is expected to decrease in affected regions, alongside an overall increase in brain amyloid burden.In this study, a retrospective analysis was performed on 18F-AV-45 PET and rs-fMRI data from 65 subjects (30 males, 35 females; mean age ± SD: 74 ± 7.4 years), across four clinical groups (Control, Early Mild Cognitive Impairment (EMCI), MCI, and AD). The regional BOLD signal complexity measures, or transient information (TI) values, were determined from the change in uncertainty of BOLD pattern prediction over time (i.e. block entropy growth rate).The main findings in this study included an expected regional reduction in functional complexity with increasing amyloid burden, as well as an unexpected, albeit non-significant, global increase in functional complexity with increased amyloid burden and disease progression. One third (27 out of 82) of the cortical and subcortical grey matter regions presented a significant (>90% CI) effect of brain amyloid load on BOLD signal complexity, including regions associated with disease-related dysfunction in memory, language processing, attention, behavior, somatosensory functions, and motor functions. With disease diagnosis taken into account, only the EMCI group indicated a decrease in global BOLD signal complexity with increased brain amyloid load.Analysis of rs-BOLD signal complexity has the potential to provide a more accurate representation of disease state than current amyloid plaque or structural measures, as well as identify the regions altered by disease pathology. Based on the reduced signal complexity reported in regions previously linked to disease pathology (precuneus/posterior cingulate, lateral temporal lobe, and frontal regions), further study of this novel metric is advised. With validation, the BOLD complexity analysis metric can be attuned to use as a cognitive biomarker in the clinical setting, potentially improving disease diagnosis, treatment monitoring, and evaluation of future treatment options.

Mitochondria are essential for promoting cell survival and growth through aerobic metabolism and energy production. Mitochondrial function is typically analyzed using mitochondria freshly isolated from tissues and cells because they yield tightly coupled mitochondria, whereas those from frozen tissue can consist of broken mitochondria and membrane fragments. A method, utilizing a well-characterized cryoprotectant such as dimethyl sulfoxide (DMSO), is described. Such mitochondria show preserved structure and function that presents us with a possible strategy to considerably expand the time-frame and the range of biochemical, molecular and metabolic studies that can be performed without the constraints of mitochondrial longevity ex vivo. Mitochondrial dysfunction is implicated in Alzheimer’s disease (AD) mainly through oxidative stress and altered metabolism. Mitochondria are isolated from post-mortem brain samples from selective regions of AD and control patients and, utilizing the cryopreservation strategy, analyzed for respiration and oxidative damage. While we did not observe increases in free radicals, we did observe decreased respiration and increases in oxidative damage markers in AD patients, suggesting a role for oxidative stress in mitochondrial dysfunction. While in the mitochondria, calcium (Ca2+) increases free radical generation by processes not completely understood. A new isoform of nitric oxide synthase (mtNOS) has been isolated and localized to mitochondria; though its existence and physiological role is debated. Nitric oxide synthase (NOS), when activated by Ca2+, produces nitric oxide (NO•) that can interact with ROS producing various reactive nitrogen species (RNS). These highly reactive radical species can damage DNA, proteins and lipids, ultimately resulting in cell death via apoptosis or necrosis. The current research is aimed at understanding the role of Ca2+ and NOS in oxidative stress leading to mitochondrial dysfunction. We observed a significant reduction in mitochondrial respiration with increasing doses of calcium. We also observed NOS enzyme activity and detected NOS protein in the purified mitochondrial fraction. Lastly, we were also able to show that Ca2+ increased the levels of free radicals and changes in oxidative damage markers. These results suggest the presence of NOS in mitochondria that could play a role in Ca2+ induced mitochondrial dysfunction and potentially leading to cell death as relevant to aging and neurodegenerative diseases.