The focus of this thesis is to investigate the intracellular protein-peptide complex 3-hydroxyacyl-CoA dehydrogenase (HADH), also known as ABAD (amyloid- binding alcohol dehydrogenase) and amyloid-beta peptide (Aβ). This complex has been identified in the development of Alzheimer's disease (AD), and this study tries to identify if ABAD is a useful biomarker for genetic risk profiling strategies for the early diagnosis of Alzheimer's disease, or a suitable target for disease-modifying drug development. The major aim of this project is to explore the biochemical and cellular processes activated as a result of the interaction of ABAD and Aβ. Understanding the cellular responses to these interactions could help identify important biomarkers and/or drug targets for the diagnosis or treatment of Alzheimer's disease. This study assesses the cytotoxic effects of Aβ in tissue culture and in animal models overexpressing ABAD. An in vitro cell system using SK-N-SH cells was developed for investigating the effects of ABAD expression in cells when incubated with synthetic Aβ peptide. In vitro studies confirmed ABAD to be a mitochondrial protein. There were problems with the efficiency of the synthetic Aβ peptide used, which was found to aggregate excessively. Trial of a soluble oligomeric Aβ peptide proved to be more efficient. A cell system was also developed, culturing neurospheres from murine stem cells. This proved to be a reliable system for culturing primary cells and keeping them in culture for up to 8 weeks. Cells were grown from a wild type strain and then differentiated and stained for endogenous expression of proteins. Proteomic studies were carried out with novel transgenic mouse models for AD. Seven proteins were identified with changed expression in the 2x Tg mouse model. Further immunocytochemistry of human AD brain tissue confirmed the upregulation of peroxiredoxin II and endophilin I. Both proteins could be returned to normal expression in the mouse models by peritoneal injection for two weeks of a novel peptide inhibitor to ABAD, confirming the involvement of the ABAD/Aβ complex in the increased expression of these proteins. Finally ABAD/Aβ was investigated as a possible target for AD therapy by screening with a small molecule fragment library. Only an initial screen was carried out, but several small molecule compounds were found to bind to ABAD. Further screening may produce lead compounds for a synthetic drug to inhibit the enhanced Aβ toxicity associated with the ABAD/Aβ complex.

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Alzheimer's disease (AD) is the leading cause of senile dementia and the current lack of effective treatments provides a strong incentive for an improved therapeutic strategy. In particular, non-pharmacological treatments, such as calorie restriction (CR), were shown to be useful in delaying or preventing AD onset. This has given insight into the molecular pathways involved. Sirtuins (SIRTl-7) are evolutionary conserved enzymes that modulate life expectancy in simple organisms. In mammals, sirtuins are involved in pathways linked to age-related -.- - diseases, such as AD. In particular, they seem to be the molecular effectors of some beneficial effects mediated by external stimuli such as CR and physical activity. This thesis aims at investigating, for the first time, the involvement of all 7 sirtuins in AD. The relevance of sirtuin gene variation to AD risk was assessed by a combined mutation screening and case-control genetic study design. Sirtuin expression levels were also assessed in a transgenic mouse model of AD (APP23 mice) and within the application of a non-pharmacological approach (environmental enrichment-EE). Finally, the possible role of SIRTl in AD pathogenesis was better elucidated through in vitro studies. Overall, in vitro studies confirmed the neuroprotective role of SIRTl activator resveratrol in etiopathological contexts related to AD. Moreover, genetic studies have highlighted SIRT2 could be involved in modulating AD etiology. In particular, a common polymorphism in SIRT2 could be a weak genetic risk factor for AD. Finally, in vivo studies confirmed the beneficial effects of an EE protocol in delaying AD onset. However, despite sirtuin involvement in other environmental paradigms, no apparent modulation was registered under EE.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the developed world. While the more common late-onset AD (LOAD) is associated with genetic and environmental risk factors, the early-onset (EOAD) form is most commonly caused by mutations in the Presenilin 1 (PSi) gene. Studying EOAD can be useful as the neuropathological hallmarks of the disease are the same as in LOAD. We have used human genomic constructs of PSi in order to investigate the role of this gene in the aetiology of AD. This method offers several advantages over cDNA over-expression studies as it allows gene expression from the entire genomic locus. The construct containing the human PSi gene was subcloned to include considerable upstream (~10 kb) and downstream (~15 kb) flanking genomic sequence. Expression levels were found to be physiologically relevant and the PSi protein was shown to undergo correct post-translational processing into PSi N-terminal and C- terminal fragments (NTF and CTF, respectively). We used this construct as the genetic background for several genetic manipulations including generation of separate mutant constructs each containing one of the following FAD mutations: M146V, Delta E9 and C410Y or the artificial proteolytically-dead D257A mutation. Clonal cell lines generated from these genomic constructs were shown to express partially or completely endoproteolytically impaired PSi proteins. In addition, the mutations were found to cause a partial to complete loss of y-secretase function with respect to Notch processing highlighting the loss of function mechanism of FAD mutations. Having engineered these mutations onto a WT-PSl-2A-Luciferase genomic construct, we expressed these in neuronal-like and PSdKO cell lines and found repression of expression with the FAD mutations tested (M146V, Delta E9 and C4i0Y). Interestingly, M146V showed a neuron-specific repression of expression highlighting an as yet unknown pathological mechanism for PSi FAD mutations. Together, these findings show that a physiologically relevant cell culture model system can be used to detect pathological mechanisms that are not detectable in an over-expressed cDNA transgenic system, which lacks the full genetic complexity of the human PSi locus.

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Amyloid-f31--42 (Af3) is considered a major mediator of the cognitive impairments that are seen in Alzheimer's disease, the leading form of dementia and a growing world-wide health problem. In vitro, Af3 treatment of hippocampal brain slices impairs long-term potentiation and enhances long- term depression, forms of synaptic plasticity, and as such serve to provide cellular correlates of this action. However, the mechanisms underlying these effects are not fully understood. In this thesis the mechanisms underlying the Aj3-mediated dysregulation of synaptic plasticity are therefore explored. Exogenously applied Aj3 was first confirmed to inhibit an NMOAR-dependent form of L TP in the hippocampus (in both acute hippocampal slices and cultured hippocampal slices) and the characteristics of the inhibition of L TP by Aj3 were examined. Next, the inhibition of L TP was found to be reliant on the activation and function of caspases, which themselves have recently been implicated in a newly characterised mechanism of L TO. Finally, evidence is provided showing that this mechanism forms part of a signalling cascade that likely reflects the cleavage and inactivation of Akt-1 by caspases, liberating GSK-3j3 from inhibition - a key L TO-signalling mechanism. These results are best explained by a mechanism whereby the exogenous application of Aj3 causes the induction of L TO signalling. The consequence of which is to inhibit LTP.

Alzheimer's disease (AD) is an incurable neurodegenerative disorder, and the most common form of dementia, characterised neuropathologically by Aβ plaques and tau tangles. Although the late-onset form of AD (LOAD) is highly heritable (-80%), genetic studies to date have only accounted for approximately half of this. Identification of the remaining risk may require new approaches. In this thesis three candidate genes/loci (CNTN2, FGA and SPARCL 1), encoding putative LOAD cerebro-spinal fluid biomarkers (Contactin-2, Fibrinogen alpha-chain and Sparc-like-1), have been subjected to complementary genetic approaches to unearth novel LOAD risk alleles. An 'LD-aware' meta-analysis of three LOAD genome-wide datasets was conducted to investigate disease association with common (MAF>5%) variation in each candidate gene. Secondly, SOliD next-generation resequencing of 150 samples (75 LOAD, 75 controls), PCR enriched for candidate loci, was conducted to identify novel rare variation. Selected variants were subsequently validated and replicated in larger series (n=1453) using TaqMan genotyping. Meta-analysis revealed linked SNPs (rs7523477, rs4951168) downstream of CNTN2 associated with LOAD (p=3x10-5, OR=1.23(1.01-1.49) n=4898). However, this has not been replicated by subsequent GWAS. Resequencing identified 28 novel rare variations over all candidate loci. A SPARCL 1 variant (located at 88451921 (hg19)) showed association with LOAD in a UK population (p=0.023, OR=1.95(1.1 0-3.46)). Replication of this association will be required in independent series. In the post-GWAS era, new approaches to identify the remaining heritability of LOAD must be embraced. Not only will the identification of new mutations conferring risk for Alzheimer's disease benefit diagnosis, it will also foster a greater understanding of disease pathways - to which treatments can be targeted.