Alzheimer’s disease (AD) is a severe age-related neurodegenerative disorder and is the most common form of dementia. Although the pathogenesis of AD remains unknown, the deterioration of the cerebrovascular system constitutes a risk factor associated with the development of the disease. Notably, brain hypoperfusion, a feature of healthy ageing brain and AD, occurs prior to the onset of cognitive decline in AD and correlates with the severity of dementia. Although there is a clear link between hypoperfusion and cognitive alterations in AD, a causal relationship remains to be established. It was hypothesised that chronic cerebral hypoperfusion leads to the accumulation of parenchymal and vascular amyloid-β (Aβ), triggering the development of vascular lesion (microinfarcts (MIs) and haemorrhages) and altering the neurovascular unit (NVU) integrity. Second to this, it was hypothesised that reductions in Aβ levels by immunotherapy targeted to amyloid in young mice, reduce amyloid levels, and prevent vascular lesions improving cognitive performance. Three studies were conducted to test these hypotheses. In the first study, the aim was to characterise age-dependent changes in amyloidrelated pathology in a transgenic mouse model (Tg-SwDI). The temporal amyloid precursor protein (APP) expression, accumulation of parenchymal and cerebrovascular Aβ and Aβ-related microglial and astrocytic activation in the cortex, hippocampus and thalamus of the Tg-SwDI mice at 3, 6 and 9 months of age was compared to wild-type controls. Significantly higher APP expression (p < 0.05), as well as Aβ aggregation (p < 0.001) as the animals aged was found in the Tg-SwDI mice in all the brain regions analysed, which was accompanied by extensive and progressive activation of microglial (p < 0.001) and astrocytic (p < 0.01) cells. These data provided a basis to design the next studies, as it was planned to induce hypoperfusion in these mice before significant Aβ deposition occurs. In the second study, the aim was to investigate the effect of hypoperfusion on Aβ dynamics and subsequently, to study the contribution of hypoperfusion and Aβ pathology to the development of MIs and haemorrhages, and to the potential alteration of astrocyte and tight junction (TJ) integrity. To address this, mild chronic cerebral hypoperfusion was induced in Tg-SwDI and wild-type mice by bilateral common carotid stenosis for 1 and 3 months. A significant increase in soluble Aβ40/42 levels was initially found after 1 month of hypoperfusion in the parenchyma (Aβ40, p = 0.0239; Aβ42 p = 0.0198) in parallel with elevated APP levels and APP proteolytic cleavage products (p < 0.05). Thereafter, following 3 months, a significant increase in insoluble Aβ40/42 levels was determined in the parenchyma (Aβ40, p = 0.0024; Aβ42 p = 0.008) and vasculature (Aβ40, p = 0.0046; Aβ42 p = 0.0118) of Tg-SwDI mice. There was no change in the levels of Aβ co-localised to vessels following 1 month of hypoperfusion; however Aβ levels were significantly increased in cerebral vessels after 3 months (p = 0.0483). The proportion of Aβ containing vessels was significantly higher in the small vessels of the hypoperfused animals compared to sham mice (p < 0.05). MIs associated with microglial proliferation were present in the Tg-SwDI mice and the burden was exacerbated by hypoperfusion at 1 and 3 months (p < 0.05). Significantly higher levels of NADPH Oxidase-2 (NOX2) were found in the transgenic mice compared to the wild-type controls at both time-points analysed (p < 0.05), and this was exacerbated after 1 month of hypoperfusion in the Tg-SwDI mice (p < 0.05). There was a positive correlation between NOX2 and soluble parenchymal Aβ levels (r = 0.6643, p = 0.0019). A minimal effect on the development of haemorrhages at these time-points was observed. In parallel to this, astrocyte activation was significantly higher in the Tg-SwDI mice compared to the wild-type controls at both time-points studied (p < 0.05); however, no effect of hypoperfusion was observed. Also, significantly higher levels of aquaporin-4 (AQP4) in the Tg-SwDI mice compared to the wild-type controls following 1 month of hypoperfusion were found (p < 0.001). There was a positive correlation between AQP4 and soluble parenchymal Aβ levels (r = 0.4735, p = 0.0095). Claudin-5 levels were significantly higher in the Tg-SwDI mice compared to the wild-type controls at both time-points analysed (p < 0.0001), and this was exacerbated following 1 month of hypoperfusion in the transgenic model (p < 0.05). A positive correlation between claudin-5 and vascular Aβ levels was observed (r = 0.6113, p = 0.0004). Together, these data suggest a synergistic contribution of amyloid and hypoperfusion pathologies to the tissue damage and implicate a role of oxidative stress and inflammation. In the third study, the aim was to determine the effects of passive amyloid immunisation on Aβ levels, development of MIs and haemorrhages and behavioural performance in the Tg-SwDI mice. To address this, the mice underwent weekly intraperitoneal injections with either 3D6 or 10D5 antibodies during 3 months. Although there were no significant changes between control and 10D5/3D6 treated mice in amyloid levels, appearance of MIs and cognitive performance, it was noted that there was a trend towards a reduction in amyloid levels and MI area in the 10D5/3D6 treated mice compared to the control animals. Furthermore, there was no evidence of microhaemorrhages in response to the immunisation. These results demonstrate that Aβ immunotherapy with the antibodies 3D6 and 10D5 may potentially decrease parenchymal and vascular amyloid accumulation, reducing the appearance of MIs and notably without triggering the development of microhaemorrhages. Collectively, the findings presented in the current thesis demonstrate that chronic cerebral hypoperfusion increases parenchymal and vascular Aβ levels and point towards a mechanism in which the cascade of events including inflammation and oxidative stress, triggered synergistically by hypoperfusion and Aβ, resulted in the widespread development of MIs and NVU changes which may further induce the alteration of cognition networks. A mixed therapy, aimed at improving cerebrovascular health and targeting the accumulation of Aβ, represents a promising strategy to prevent neurodegenerative processes and further cognitive decline in AD.

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Mass Spectrometry (MS) and Ion Mobility - Mass Spectrometry (IM-MS) can be used to investigate protein structure and dynamics and are ideally positioned to study intrinsically disordered and amyloidogenic proteins, whose diverse conformational space and/or oligomeric state is hard to track accurately. This thesis uses hybrid MS approaches including IM-MS, Cross-linking IM-MS and ECD-FT-ICR MS to probe the structure of alpha-Synuclein and Amyloid-beta (Abeta). For alpha-Synuclein, the effect of solution pH and ionisation polarity on the species observed by MS and IM-MS is investigated. Conformational families observed by Cross-linking IM-MS provides a link between the solution and gas phase structures of alpha-Synuclein observed here and our data correlates with that reported by other groups. MS, IM-MS and HDX-MS are used to probe alpha-Synuclein during the early stages of aggregation. A specific aggregation competent conformer is not observed suggesting that the solution constituents remain conformationally dynamic. We observe shifts in the species observed by MS and IM-MS between samples and our data contributes to an array of conflicting structural studies indicating that alpha-Synuclein adopts a diverse range of species with significant variation. For Abeta(1-42) and Abeta(1-40) Collision Induced Unfolding and ETD/ETcaD demonstrate that Abeta(1-42) adopts a compact conformation bound by intramolecular interactions. Changes to the Abeta(1-42) and Abeta(1-40) ATDs following SID are correlated to known structure influencing intermolecular interactions and demonstrate the large structural difference between Abeta(1-42) and Abeta(1-40) despite differing by only two C-terminal amino acids. IM-MS is used to classify the mode of action of anti-aggregation drug candidates on Abeta(1-42). The anti-aggregation capacity of the retro-inverso peptide, RI-OR2 is shown to result from inducing the compaction or extension of Abeta(1-42), preventing the adoption of an aggregation competent structure. In contrast, the flavonoid Rutin is shown to act solely through inducing Abeta(1-42) compaction. This thesis demonstrates the power of MS based methods to investigate the diverse range of structures of intrinsically disordered aggregating proteins implicated in disease.

Dementia, the most common cause of which is Alzheimer's disease (AD), currently affects 850,000 people in the UK, a figure set to rise to over 1 million by 2025. There is currently no disease modifying therapy available to slow or halt this progressive disease. Current understanding of AD implicates the neurotoxic amyloid-β (Aβ) peptide as the primary initiator in a cascade of events leading to the neuronal cell death and brain atrophy associated with the disease. Therefore, inhibiting the production or enhancing the clearance of Aβ within the brain has become a major target for the production of disease modifying therapeutics. Aβ is produced by brain cells through the sequential proteolytic cleavage of a larger transmembrane protein known as the amyloid precursor protein (APP) by β- and γ-secretases. Several aspects of APP physiology can influence its proteolysis, and thus Aβ production, including the isoform of APP which is expressed, its trafficking and subcellular location and its physical interactions with other proteins in the cellular environment. Here we have investigated the influence of subcellular trafficking and location and protein-protein interactions on the differential proteolysis of two APP isoforms, APP695 and APP751 in a neuroblastoma cell line. We have shown that APP751 undergoes less amyloidogenic proteolysis than APP695 and that retention within the early secretory pathway may contribute to this difference. APP751 shows higher co-localisation to the trans-Golgi network than APP695 in immunofluorescence microscopy studies, while addition of a mutation which causes APP proteolysis in the secretory pathway reduces the large difference in amyloidogenic proteolysis of these two isoforms. Targeting APP endocytosis from the cell surface, thought to be a key determinant in Aβ generation, effects APP isoform proteolysis and Aβ production to a similar extent in both the APP isoforms suggesting differences in proteolysis occur before this trafficking event. We also show by immunoblot analysis that the APP isoforms may be differentially cleaved by proteases other than β- and γ-secretase to produce recently identified proteolytic fragments. Using a liquid chromatography - tandem mass spectrometry approach coupled to prior stable isotope labelling of amino acids in cell culture (SILAC), we have identified the interactomes of the two APP isoforms in our model system. Gene ontology analysis identified enrichment of nuclear and mitochondrial proteins specifically in the APP695 interactome. Using siRNA mediated protein knockdown, we have shown interactions with Fe65 and ataxin-10 specifically influence Aβ generation from the APP695 isoform. Fe65 alters proteolysis at the rate limiting β-secretase cleavage step, while ataxin-10 alters proteolysis by γ-secretase. Interaction with growth-associated protein 43 specifically influences Aβ generation from the APP751 isoform, altering proteolysis at the γ-secretase step. Finally we have shown that recently discovered familial AD-linked mutation and protective mutation within the Aβ region of the APP protein have consistent effects on APP proteolysis in both the APP isoforms.

The aim of the thesis project was to examine what form(s) of Amyloid beta (Aβ) (25-­‐35) peptide interact with phospholipids in vitro and the implications of this for the mechanism of Alzheimer’s Diseases (AD). The mechanism of AD is thought to involve protein folding and misfolding. An increasing amount of evidence has shown that protein misfolding plays an important role in the biological and pathological processes of AD. Although seen as the biomedical markers of those diseases, the roles of amyloid aggregates themselves are still not fully understood. Whether the aggregates, or the monomer, or some other intermediates of Aβ cause AD is still unknown. In order to investigate the membrane-­‐interaction of Aβ and its implications for AD, two forms of Aβ, namely levorotary and dextrorotary (L-­‐ and D-­‐) Aβ isomers were used. Evidence has shown that L-­‐ and D-­‐ peptide can each form aggregates in a humid environment. However, when mixed together, L-­‐ and D-­‐ peptides tend not to form any aggregates. Using the mixtures of L-­‐ and D-­‐ peptides at different proportions and as well as using L-­‐ and D-­‐ alone can help us to determine the toxic form of Aβ. Phospholipids have been used to mimic membrane bilayers. Biological membranes in vivo are a complicated system. They contain three types of lipids, namely phospholipids, glycolipids, and steroids. Different types of cells and different membranes have different proportions of those lipids. Studying the interaction between Aβ and membranes in vivo can be extremely difficult. Artificial membranes, which only contains one kind of lipids, on the other hand, are a useful tool for the study of molecular interactions. Phospholipids are the most abundant type of membrane lipid and thus that can be seen as representative of cell membranes. The interactions of Aβ and different kinds of phospholipids have been investigated in this project. This thesis discusses the secondary structure of Aβ in different environment, the interaction between Aβ and phospholipids at the air-­‐water surface, and the location of Aβ in membranes during the interaction. The study provides useful information of the mechanisms and the origin of AD. At the end of the thesis, a discussion chapter analyses the difficulties of studying Aβ and AD and the potentials and inadequacies of this research.

Alzheimer's disease (AD) is characterised by memory loss, insidious cognitive decline, profound neurodegeneration, and the extracellular accumulation of amyloid-beta (Aβ) peptide in senile plaques and intracellular accumulation of tau in neurofibrillary tangles. Synaptic dysfunction and loss is the strongest pathological correlate of cognitive decline in AD with increasing evidence implicating neuropathological forms of both amyloid-beta and tau protein in this process. A large amount of evidence suggests that oligomeric forms of Aβ, associated with senile plaques, are toxic to synapses but the precise localisation of Aβ and which forms are synaptotoxic remain unknown. Using the high-resolution technique, array tomography, this thesis characterised the synaptic localisation of different forms of Aβ oligomers in a mouse model of amyloidopathy. These results show that different oligomeric Aβ species are present in both presynapses and postsynapses. This study highlights the potential of array tomography for rapid testing of aggregation state specific Aβ antibodies in brain tissue. Following these results, the presence of tau at synapses was examined. Despite the knowledge that tau spreads through defined synaptic circuits, it is currently unknown whether synapse loss occurs before the accumulation of tau or as a consequence. To address this, array tomography was used to examine a mouse model in which mutant P301L human tau is expressed primarily in the entorhinal cortex (rTgTauEC). It has previously been shown that rTgTauEC mice exhibit neuronal loss in the entorhinal cortex and synapse density loss in the middle molecular layer (MML) of the dentate gyrus at 24 months of age. The density of tau-expressing and total presynapses, and the spread of tau into the postsynapse in the MML of 3-6, 9, and 18 month old mice were examined. No loss of synapse density was observed in the MML up to 18 months of age, even in axons expressing tau. Despite the maintenance of synapse density, we see spread of human tau from presynaptic terminals to postsynaptic compartments in the MML at very early ages. This indicates that the spread of tau through neural circuits is not due to the degeneration of axon terminals and is an early feature of the disease process. Following examination of both synaptic amyloid-beta and tau in separate models, this thesis then examined how these two proteins may be synergistically working together to drive synaptic pathology. To investigate this a novel mouse model was used in which amyloid-beta deposits are present in combination with non-mutated human tau expression (APP/PS1 + hTau). These results suggested that the addition of human tau expression does not increase plaque associated synapse loss, neither does it increase the proportion of synapses colocalising with amyloid-beta. Similarly the presence of human tau at individual postsynapses was not enhanced in the presence of oligomeric Aβ. Surprisingly, intact long-term recognition memory was observed in APP/PS1 + hTau mice. However a hyperactive phenotype was detected in these mice that could be prevented upon tau suppression. This suggests a synergistic relationship may exist in the presentation of this phenotype. Finally in the last part of this thesis, synapses from post-mortem human Alzheimer's disease and age-matched controls were investigated. It has previously been suggested that both amyloid-beta and tau can interfere with mitochondrial transport to the synapse and mitochondrial function. For this reason the presence of synaptic mitochondria at both the presynapse and postsynapse was determined in order to investigate any alteration in the diseased state. A reduction in the proportion of presynapses with multiple mitochondria present was detected in anterior/posterior transverse temporal cortex (BA41/42). This was not observed in dorsolateral prefrontal cortex (BA46), suggesting either a selective vulnerability of the former brain region or a selective resistance of the latter brain region, to mitochondrial depletion at the synapse. The findings presented in this thesis demonstrate that when investigated in isolation, pathological forms of amyloid-beta are present at a subset of synapses where they may contribute to toxicity, whilst the spread of tau protein is an early feature of the disease process and occurs prior to overt synapse loss. This thesis also explores the proposed synergistic relationship between amyloid-beta and tau using a novel mouse model and human post-mortem brain tissue. Since these two proteins both have been implicated in synaptic dysfunction, investigating Aβ and tau in new mouse models and human brain tissue will be instrumental in furthering our understanding of mechanisms and features of synaptotoxicity that could be important therapeutic targets.

Mechanobiology is an emerging field that aims to understand how physical forces regulate cell function, morphology, and development. Cells interpret forces, such as the deformation of the membrane to encapsulate a particle, or the rigidity of the extracellular matrix (ECM), and make decisions about cell adhesion, motility, and differentiation. These cell-ECM interactions are important to maintaining homeostasis, and the disruption of this interface has pathological consequences. Common diseases, such as Alzheimer’s disease, cancer, and atherosclerosis each arise, in part, from an abnormality in the mechanotransduction pathway. Hence, understanding the contribution of this pathway and the role of the ECM in cell function, proves to be a useful tool in improving drug targeting and understanding disease progression. While size, shape and surface chemistry of nanoparticle uptake has been extensively studied, varying the particle mechanics can also be a useful design strategy to manipulate particles and improve uptake and targeting. Using model polystyrene-co-N-isopropylacrylamide (pS-co-NIPAM) particles, with varying elastic moduli, it was observed that as the particles became stiffer, there was a subsequent decrease in bound/internalized particles for phagocytic RAW264.7 macrophage and non-phagocytic HepG2 hepatoma carcinoma cells, showing that both of these cell types are sensitive to particle mechanics, even in a higher stiffness regime (MPa). ECM mechanics have recently been implicated in tissue stiffness changes that precede and drive disease development. Recent research has started looking into these effects in the progression of neurodegenerative diseases. This research found that the elasticity of the brain becomes softer with aging, and even softer in patients with AD. Analogous to the pS-co-NIPAM studies, this tissue softening could have implications on amyloid-beta endocytosis as well as neuron dystrophy in response to the peptide. Understanding the role of the ECM in the progression of AD in vitro could provide a better approach to determine an in vivo mechanism behind Alzheimer’s disease pathology. In order to mimic a softer ECM substrate, SH-SY5Y neuroblastoma and human primary neurons were plated on 2-D polyacrylamide and 3-D collagen gels with varying stiffness ranging from 0.15-25kPa. Both cell types grown using these substrates show a sensitivity to their ECM environment, and display an increase in cell spreading and the number of F-actin stress fibers with an increase in substrate rigidity. Moreover, the extent of Aβ internalization and aggregate production increased with ECM stiffness for SH-SY5Y neuroblastoma. Intracellular Aβ processing remains a central question to understanding the early-stage events in AD pathogenesis. As the ECM can modify Aβ endocytosis and aggregation, the ECM is likely influencing downstream neurotoxic effects of AD. Despite an increase in the plaque production on the soft substrates, both SH-SY5Y neuroblastoma and primary neurons showed a decreased toxicity to Aβ with decreasing substrate stiffness. This decrease in toxicity is associated with cytoskeletal actin remodeling, as cells plated on plastic, but pretreated with cytochalasin D displayed a recovery in viability in response to the oligomeric species. The softening of the ECM initiates actin cytoskeletal depolymerization, as a protective mechanism against neuronal loss and AD progression. This work demonstrates that the ECM impacts Aβ endocytosis and aggregation, and the ECM prompts neuroprotective actin reorganization against the neurotoxic effects of AD. Further, it is demonstrated the biophysical role of ECM stiffness in modifying Aβ internalization, plaque production, and toxicity offers an improved in vitro model of critical AD components. By better understanding the cytoskeletal reorganization triggered by a softening ECM, potential novel avenues of therapeutic intervention could later be determined to stop the progression of the disease.

Alzheimer’s disease (AD), identified over 100 years ago and intensively studied since the 1970s, has no effective treatments or mechanistic understanding of the underlying neurodegenerative process. Most investigators believe accumulation or aggregation of amyloid beta (Ab) proteins plays a causative role. Aβ peptides (~39-43 residues) are generated by proteolysis of the transmembrane protein APP. One reason we know so little about AD is an incomplete understanding of the cellular mechanisms responsible for Ab proteotoxicity. Human ES and iPSC models of AD are recent additions to many other models used to investigate these mechanisms. AD, however is a chronic progressive condition of old age and cultured neurons may not live long enough to model what goes wrong in neurons from AD patients. In my research, I used hESCs which directly express Ab peptides thus avoiding the time it takes to process APP. One App allele in H9 hESCs was previously edited using TALEN. A homologous recombination cassette coding directly for a secretory form of either Ab1-42 or Ab1-40 and containing a stop codon, was inserted into the first exon of App upstream of the normal translational start site. I used multiple independently isolated clones of edited cells with 3 genotypes: App/App (unedited), App/Aβ1-40 and App/Aβ1-42. Expression of Ab from edited alleles was confirmed by qRT-PCR using primers specific for the edit. I first sought to establish if editing changed any aspects of neuronal differentiation in culture. All 3 genotypes have similar embryoid body (EB) development, and similar numbers and sizes of neuronal clusters (NC) up to 34 days after EB dissociation and neural differentiation. Immunostaining of neuronal markers, NeuN and DCX (doublecortin), likewise revealed no difference among edited and unedited cells, suggesting that the edits do not affect the ability of my stem cells to differentiate into neurons. I next measured accumulation of aggregated Ab using an aggregate specific antibody, 7A1a. Data at 34-days post EB dissociation indicates NCs in the Aβ1-42 edited cells accumulate significantly more aggregates relative to either unedited or Ab1-40 edited lines, a result consistent with the increased ability for Ab1-42 to form aggregates. Aβ aggregates also appear to be concentrated around fragmented nuclei within neuronal clusters suggesting that intracellular accumulation may play a key role in proteotoxicity. Additionally, I observed a significant decrease in the number of synapsin1 puncta, a marker of synapses, another feature of AD. I documented a nearly 3-fold greater neuronal cell death in both the Aβ1-40 and Aβ1-42 neurons at 70 days after differentiation. RNA sequencing data also shows independently isolated clones group together and show differential expression of genes related to memory and neuronal cell death. The early presence of Aβaggregation and subsequent cell death is in line with the chronic and progressive nature of AD and this is the first known model to exhibit a neurodegenerative phenotype. These isogenic cell lines thus appear to be useful to screen for therapeutics that may prevent or slow Ab1-42 dependent neurodegeneration and a tool to investigate Ab-dependent mechanisms with relevance to AD.

ABSTRACT Alzheimer's disease (AD) is the most common progressive age related dementia and the fourth major cause of mortality in the elderly in the United States. AD is pathologically characterized by deposition of amyloid beta (Aβ) plaques in the brain parenchyma and neurofibrillary tangles (NFTs) within the neuronal soma. While pharmacological targets have been discovered, current strategies for the symptomatic or disease-modifying treatment of AD do not significantly slow or halt the underlying pathological progression of the disease. Consequently, more effective treatment is needed. One possibility for amelioration is using human umbilical cord blood cell (HUCBC) therapy. HUCBCs comprise a population of hematopoietic stem and progenitor cells. During recent years, functional recovery has been observed from the use of HUCBCs in pre-clinical animal models of brain and spinal cord injuries. Thus, modulation by cell therapy, specifically HUCBCs, may be a suitable treatment for AD and other models because of the observed cognitive and behavioral improvements. The studies presented in this dissertation centers on the suitability of using HUBCs as a potential treatment for AD. Expanding on this, the aims of the study sought to: (I) Investigate bio-distribution of HUCBC transplantation in PSAPP mice, (II) Characterize efficacy and determine therapeutic outcome of HUCBC following short and long term multi injections at early and late disease stages in PSAPP mice and (III) Determine AD-like pathological and cognitive changes associated with multiple HUCBC-derived monocyte (CD14) injections in PSAPP mice. Thus the findings of this work evolved from experiments that characterized the effects of low-dose infusions of HUCBC and HUCBC-derived monocytes into 6 month old Presenilin 1/Amyloid Precursor Protein (PSAPP) plaque-developing transgenic AD mice. Treated mice were studied using standard behavioral tests to determine the effects of infusion on the multiple cognitive domains affected by AD, followed by biochemical and histological analyses that included Aβ load and amyloid precursor protein (APP) processing. Specifically, PSAPP mice and their wild-type (WT) littermates were treated monthly with a peripheral HUCBC infusion over a period of 6 and 10 months, followed by cognitive and motor evaluation. Additionally, based on reports that tumor cells can originate from stem cells present in HUCB, we further examined whether monocytes purified from HUCBCs would have a similar significant effect on the reduction of AD-like pathology in PSAPP mice. HUCB cells homed into tissues including the brain. The principal finding was significant reduction in Aβ levels and β–amyloid plaques following low-dose infusions of both HUCBC– derived mononuclear cells as well as HUCBC-derived monocytes, with the monocytes providing a stronger effect. Results further demonstrated that HUCBC and HUCBC– derived monocyte infusion could improve memory function and locomotor ability in treated PSAPP mice. A possible reason for behavioral improvements in these animals may be the significant reduction in both Aβ levels and plaque load. This study also identified significant reduction in microglial activation and astrocytosis, both of which can contribute to AD pathology. In conclusion, our data suggest that it might be the HUCBC–derived monocytic population rather than stem cells that are responsible for the reduction in AD pathology.

The neurodegenerative disorders Alzheimer??s Disease (AD) and Parkinson??s Disease (PD) are characterised by the accumulation of misfolded amyloid beta 1-42 peptide (Aβ1-42) or α-synuclein, respectively. In both cases, there is extensive evidence to support a central role for these aggregation-prone molecules in the progression of disease pathology. However, the precise mechanisms through which Aβ1-42 and α-synuclein contribute to neurodegeneration remain unclear. Organismal, cellular and in vitro models are under development to allow elucidation of these mechanisms. A cellular system for the study of intracellular Aβ1-42 misfolding and localisation was developed, based on expression of an Aβ1-42-GFP fusion protein in the model eukaryote Saccharomyces cerevisiae. This system relies on the known inverse relationship between GFP fluorescence, and the propensity to misfold of an N-terminal fusion domain. To discover cellular processes that may affect the misfolding and localisation of intracellular Aβ1-42, the Aβ1-42-GFP reporter was transformed into the S. cerevisiae genome deletion mutant collection and screened for fluorescence. 94 deletion mutants exhibited increased Aβ1-42-GFP fluorescence, indicative of altered Aβ1-42 misfolding. These mutants were involved in a number of cellular processes with suspected relationships to AD, including the tricarboxylic acid cycle, chromatin remodelling and phospholipid metabolism. Detailed examination of mutants involved in phosphatidylcholine synthesis revealed the potential for phospholipid composition to influence the intracellular aggregation and localisation of Aβ1-42. In addition, an existing S. cerevisiae model of α-synuclein pathobiology was extended to study the effects of compounds that have been hypothesized to be environmental risk factors leading to increased risk of developing PD. Exposure of cells to aluminium, dieldrin and compounds generating reactive oxygen species enhanced the toxicity of α- synuclein expression, supporting suggested roles for these agents in the onset and development of PD. Expression of α-synuclein-GFP in phosphatidylcholine synthesis mutants identified in the Aβ1-42-GFP fluorescence screen resulted in dramatic alteration of α-synuclein localisation, indicating a common involvement of phospholipid metabolism and composition in modulating the behaviours of these two aggregation-prone proteins.