Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 100-123).

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

The cellular prion protein (PrP) has been shown to act as a receptor for soluble oligomers of amyloid-beta (Aβ), an ~4 kDa amyloidogenic peptide that is found in neuritic plaques that are a pathological hallmark of Alzheimer’s disease (AD). Oligomeric forms of the Aβ peptide are thought to be synaptotoxic, and have been shown to produce PrP-dependent dendritic spine loss, suppression of long term potentiation (LTP), and behavioral changes in mouse models of AD. However, the specific molecular interactions between PrP and Aβ have not been fully characterized. In this work, we conducted a robust examination of the kinetic processes leading to Aβ fibril formation, and present evidence that PrP significantly inhibits Aβ polymerization. Using established mathematical models of polymerization kinetics, we show that inhibition is based on binding between PrP and the ends of Aβ filaments, an interaction that blocks elongation. To support these results, we conducted multiple binding assays to show that PrP binds to monomers of Aβ with low affinity, oligomers with intermediate affinity, and to fibrils with high affinity. These results extend upon previous studies, which have focused only on the interaction between oligomeric Aβ and PrP. To better understand the molecular interactions required for binding and inhibition of polymerization, we performed assays with a series of PrP deletion mutants, which revealed that low-affinity binding to Aβ monomer is dependent on the presence of the C-terminal domain of PrP. This domain is also required for Aβ polymerization inhibition. Based on our results, we propose a model in which the unstructured N-terminal domain of PrP binds to the ends of Aβ fibrils, while the C-terminal domain interrupts the docking of new monomers to fibril ends, in part through competing for similar binding sites. This study provides an important contribution to our understanding of the PrP-Aβ interaction that leads to synaptoxicity.

Yes / Astrocytes play a fundamental role in maintaining the health and function of the central nervous system. Increasing evidence indicates that astrocytes undergo both cellular and molecular changes at an early stage in neurological diseases, including Alzheimer’s disease. These changes may reflect a change from a neuroprotective to a neurotoxic phenotype. Given the lack of current disease modifying therapies for Alzheimer’s disease, astrocytes have become an interesting and viable target for therapeutic intervention. The astrocyte transport system covers a diverse array of proteins involved in metabolic support, neurotransmission and synaptic architecture. Therefore, specific targeting of individual transporter families has the potential to suppress neurodegeneration, a characteristic hallmark of Alzheimer’s disease. A small number of the four hundred transporter superfamilies’ are expressed in astrocytes, with evidence highlighting a fraction of these are implicated in Alzheimer’s disease. Here we review the current evidence for six astrocytic transporter subfamilies involved in Alzheimer’s disease, as reported in both animal and human studies. This review confirms that astrocytes are indeed a viable target, highlights the complexities of studying astrocytes and provides future directives to exploit the potential of astrocytes in tackling Alzheimer’s disease. / BBSRC, Alzheimer's Society, Motor Neuron Disease Association

No description available.

No description available.

Sporadic Inclusion Body Myositis (sIBM) is an inflammatory muscle disease that strikes individuals at random and accounts for approximately 1/3 of all idiopathic inflammatory myopathies. It is characterized by progressive weakness of distal and proximal muscles and is the most common muscle disorder in individuals over 50 years of age. Currently, there is no known cause, cure, or enduring treatment for sIBM, although a number of theories as to its cause have been proposed. One theory proposes that activation of the inflammatory/ immune response is the primary trigger resulting in muscle degeneration and protein abnormalities, while an alternative theory suggests that sIBM is a degenerative muscle disease with abnormal pathogenic protein accumulation, in particular Abeta, being a primary cause that triggers an inflammatory/ immune response. Mitochondrial abnormalities have been observed in skeletal muscle from patients diagnosed with the disease, however the role of the mitochondria in disease pathology is still unclear. The aim of this dissertation was to evaluate: 1) the role of the mitochondria in the development of sIBM and 2) the role of amyloid beta on mitochondrial function in skeletal muscle. A better understanding of the role of the mitochondria in the development of sIBM may help to identify novel prevention and/ or treatment strategies. / Ph. D.

Amyloid plaques, a neuropathological hallmark of Alzheimer’s disease (AD), are largely composed of amyloid beta (Aβ) peptide, derived from cleavage of amyloid precursor protein (APP) by β- and γ-secretase. The endosome is increasingly recognized as an important crossroads for APP and the secretases, with major implications for APP processing and amyloidogenesis. Amongst various posttranslational modifications affecting APP, ubiquitination of cytodomain lysines may represent a key signal controlling endosomal sorting. Here, we show that substitution of APP COOH-terminal lysines with arginines disrupts APP ubiquitination, though the pool of ubiquitinated APP is small or transient. Nonetheless, this small deficiency in ubiquitination can have a significant impact on APP, such that the number of lysines mutated trends toward an increase in APP metabolism. An APP mutant lacking all COOH-terminal lysines undergoes the most pronounced increase in processing, leading to accumulation of both secreted and intracellular Aβ40, without change in Aβ42. This phenotype is abolished by artificial ubiquitination of APP using rapalog-mediated proximity inducers. Lack of APP COOH-terminal lysines does not affect APP endocytosis, but leads to a redistribution of APP from endosomal intraluminal vesicles (ILVs) to the endosomal limiting membrane, with subsequent decrease in APP COOH-terminal fragment (CTF) content of secreted exosomes, but minimal effects on APP lysosomal degradation. Both the secreted and intracellular increase in Aβ40 is abolished by depletion of presenilin 2 (PSEN2), recently shown to be enriched on the endosomal limiting membrane compared to presenilin 1 (PSEN1). In a separate set of studies, we found that a familial AD mutant, L723P, which occurs immediately next to a string of three lysines in the juxtamembrane region, behaves more similarly to other FAD-causing mutations. APP L723P exhibits a selective increase in Aβ42, and a delay in degradation, but no change in exosomal content, despite some missorting to the endosomal limiting membrane. Our findings demonstrate that ubiquitin can act as a signal for endosomal sorting at five lysines in the APP cytodomain, disruption of which prevents sequestration of APP in ILVs and results in the processing of a larger pool of APP-CTF by PSEN2 on the endosomal membrane.

Leung Man-hin. / Thesis submitted in: August 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 119-133). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgement --- p.iv / Abbreviations --- p.v / Figure List --- p.vii / Chapter Chapter 1: --- General introduction / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Pathogenesis of Alzheimer's disease / Chapter 1.2.1 --- Amyloid cascade hypothesis --- p.2 / Chapter 1.2.2 --- Tauopathy --- p.5 / Chapter 1.3 --- The amyloid precursor proteins / Chapter 1.3.1 --- Structure of amyloid precursor proteins and related peptides --- p.5 / Chapter 1.3.2 --- Amyloid precursor protein mutations --- p.6 / Chapter 1.3.3 --- Amyloid precursor protein processing --- p.10 / Chapter 1.3.4 --- Physiological roles of APP --- p.12 / Chapter 1.3.5 --- The pathophysiological role of Ap --- p.17 / Chapter 1.3.6 --- The pathophysiological role of APP-CTF --- p.20 / Chapter 1.4 --- The role of proteases in amyloid precursor protein processing / Chapter 1.4.1 --- α-secretase and p-secretase --- p.22 / Chapter 1.4.2 --- γ-secretase complex --- p.30 / Chapter 1.4.3 --- Caspases --- p.36 / Chapter 1.4.4 --- Kallikrein-like proteases --- p.37 / Chapter 1.5 --- Objective of the present study --- p.40 / Chapter Chapter 2: --- Materials and methods / Chapter 2.1 --- Experimental procedure / Chapter 2.1.1 --- Plasmid construction --- p.42 / Chapter 2.1.2 --- "DNA purification, ligation and restriction enzyme digestion" --- p.44 / Chapter 2.1.3 --- Competent cell preparation --- p.47 / Chapter 2.1.4 --- Transformation --- p.47 / Chapter 2.1.5 --- Plasmid miniprep --- p.48 / Chapter 2.1.6 --- Prokaryotic expression of neurosin --- p.49 / Chapter 2.1.7 --- SDS-PAGE --- p.51 / Chapter 2.1.8 --- Protein sample preparation --- p.51 / Chapter 2.1.9 --- Western Blot --- p.52 / Chapter 2.1.10 --- Immobilized metal affinity chromatography --- p.54 / Chapter 2.1.11 --- Enzyme assay --- p.55 / Chapter 2.1.12 --- Cell culture and transfection --- p.56 / Chapter 2.1.13 --- Live cell imaging --- p.57 / Chapter 2.2 --- Materials --- p.59 / Chapter Chapter 3 : --- Results / Chapter 3.1 --- Recombinant expression and characterization of neurosin / Chapter 3.1.1 --- Construction of neurosin prokaryotic expression vectors --- p.62 / Chapter 3.1.2 --- Prokaryotic expression of neurosin --- p.64 / Chapter 3.1.3 --- Neurosin was expressed as inclusion bodies --- p.68 / Chapter 3.1.4 --- Co-expression of molecular chaperones with neurosin --- p.70 / Chapter 3.1.5 --- Purification of recombinant neurosin by IMAC --- p.76 / Chapter 3.1.6 --- Enzyme assay --- p.81 / Chapter 3.2 --- Effect of neurosin on APP processing in neuronal cells / Chapter 3.2.1 --- Generation of APP constructs --- p.84 / Chapter 3.2.2 --- Expression of APP in mammalian cultures --- p.89 / Chapter 3.2.3 --- Cellular localization of APP and its processing products --- p.96 / Chapter 3.2.4 --- The role of over-expression of neurosin on APP processing in B103 cells --- p.101 / Chapter Chapter 4: --- Discussion / Chapter 4.1 --- Discussion of neurosin expression --- p.103 / Chapter 4.2 --- Discussion of APP cell model --- p.109 / Chapter 4.3 --- Conclusion --- p.119 / References --- p.121 / Appendix / Chapter A. --- Tables on primers used --- p.138 / Chapter B. --- Plasmid maps --- p.139 / Chapter C. --- Raw data on the DNA sequencing --- p.141

Alzheimer's disease (AD) is characterized by the presence of aggregates of amyloid beta (Aβ) in senile plaques and tau in neurofibrillary tangles, as well as marked neuron and synapse loss. Of these pathological changes, synapse loss correlates most strongly with cognitive decline. Understanding the contributions of different risk factors, toxic proteins, and protein networks to synaptic dysfunction and loss is essential to understanding and one day curing this disease. Oligomeric species of both Aβ and tau are implicated in synapse, however the interaction between them requires further exploration. The first aim of this thesis was to investigate the interaction of Aβ and tau in a novel mouse model AD. In this model APP/PS1 mice were crossed with mice expressing full length wild type human tau (hTau). Expression of hTau in APP/PS1 mice increased plaque size by~50% and increased plaque-associated dystrophic neurites. However, no increase in neurite curvature, neuron loss, or synapse loss was observed in the hTau APP/PS1 animals compared with APP/PS1 alone. The underlying cause of most cases of AD is not known, however genetic risk factors have been identified, the strongest of which is the APOE e4 allele. APOE e4 is associated with increased risk of developing AD and increased rates of cognitive decline compared to the more common APOE e3 allele. The second aim of this thesis was to detect differences in the AD synaptic proteome compared with controls and to also investigate the effect of an APOE e4 allele on those changes. Unbiased label free LC-MS/ MS based proteomics of synapses isolated from human AD and control post-mortem brains of known APOE genotypes was used. Of the 1043 proteins detected in 20 synaptic preparations 17% (173) were found to differ significantly (p < 0.05, fold change >1.2) in AD compared with control. A significant sub-set of these proteins were affected by APOE e4 allele genotype. One of these was Clusterin which was not only increased in the AD synapse but further increased in cases with an APOE e4 allele. Clusterin is closely related to ApoE has also been genetically linked to AD in genome-wide association studies. Aim three was to further investigate the involvement of Clusterin at the synapse and the interaction of ApoE with Clusterin using array tomography. Array tomography confirmed an increase in Clusterin co-localization with presynapses and postsynapses in AD cases compared with controls and found a further increase in cases with an APOE e4 allele. Array tomography also found an increase in synapses which co-localized with Clusterin and Aβ together in cases with an APOE e4 allele. This implies that Clusterin is important in Aβ mediated synapse loss in AD. To further investigate the role of synapse loss in AD aim 4 of this thesis was to develop a novel human based model of Aβ mediated synapse loss. This model uses cortical neurons derived from induced pluripotent stem cells from a control individual that are challenged with Aβ extracted from brains from AD and control individuals. This model shows a significant and concentration dependent reduction in the number of synapses in response Aβ from AD brain but not to control brain extract or AD brain extract immunodepleted of Aβ. The work presented in this thesis has investigated two novel models of AD to assess the effect of known toxic proteins in AD related synapse degeneration. This work also shows that profound protein changes occur at the synapse in AD and that many of these are affected by APOE genotype. Many of these changes potentially cause or contribute to synaptic dysfunction in AD and therefore could be important for therapeutic interventions.