ABCA7 is the closest homologue of ABCA1, which is established to play a key role in reverse cholesterol transport. ABCA1 deficiency causes Tangier disease and several ABCA1 modulations have been recently associated with Alzheimer’s disease. Prior research on ABCA7 focused on its role in macrophage lipid efflux and phagocytosis, however, tissue expression studies in mice revealed that ABCA7 is highly expressed in the brain. The function of ABCA7 in the brain is unknown and the purpose of this thesis was therefore to investigate the potential function of ABCA7 in the brain and the possible implications for Alzheimer’s disease. This was achieved by using in vitro models that transiently express ABCA7 and in vivo models including ABCA7 knockout (ABCA7 KO) mice and human APP transgenic (APPSwInd Tg) mice. ABCA7 was shown to potently stimulate cholesterol efflux from cells to extracellular apoE acceptors. Additionally, apoE expression was decreased by 20% in the brains of ABCA7 KO mice compared to wild type littermate controls and by 46% in ABCA7-deficient macrophages. In vitro models that stably express human APP protein demonstrated that the transient expression of ABCA7 increased intracellular APP targeting, which led to the inhibition of APP processing and a resultant decrease in the production of Aβ. In addition, ABCA7-deficient macrophages displayed impaired capacity to endocytose and degrade Aβ oligomers. Finally, in aged APPSwInd Tg mice where abundant amyloid plaques were present, ABCA7 expression was significantly decreased by 48%. Since cholesterol homeostasis modulates APP processing, the findings of this thesis suggest that ABCA7 may have a role in maintaining cellular cholesterol homeostasis via cholesterol efflux to apoE acceptors and possibly by regulating intracellular trafficking of specific membrane proteins. In summary, ABCA7 was clearly shown in this thesis to have an effect on Aβ generation, clearance and degradation. Furthermore, ABCA7 expression was also affected by Aβ deposition in the brain. Even though more work is required to further elucidate the exact function of ABCA7, the findings presented in this thesis provide the first clues to the function of ABCA7 in the brain.

ABCA7 is the closest homologue of ABCA1, which is established to play a key role in reverse cholesterol transport. ABCA1 deficiency causes Tangier disease and several ABCA1 modulations have been recently associated with Alzheimer’s disease. Prior research on ABCA7 focused on its role in macrophage lipid efflux and phagocytosis, however, tissue expression studies in mice revealed that ABCA7 is highly expressed in the brain. The function of ABCA7 in the brain is unknown and the purpose of this thesis was therefore to investigate the potential function of ABCA7 in the brain and the possible implications for Alzheimer’s disease. This was achieved by using in vitro models that transiently express ABCA7 and in vivo models including ABCA7 knockout (ABCA7 KO) mice and human APP transgenic (APPSwInd Tg) mice. ABCA7 was shown to potently stimulate cholesterol efflux from cells to extracellular apoE acceptors. Additionally, apoE expression was decreased by 20% in the brains of ABCA7 KO mice compared to wild type littermate controls and by 46% in ABCA7-deficient macrophages. In vitro models that stably express human APP protein demonstrated that the transient expression of ABCA7 increased intracellular APP targeting, which led to the inhibition of APP processing and a resultant decrease in the production of Aβ. In addition, ABCA7-deficient macrophages displayed impaired capacity to endocytose and degrade Aβ oligomers. Finally, in aged APPSwInd Tg mice where abundant amyloid plaques were present, ABCA7 expression was significantly decreased by 48%. Since cholesterol homeostasis modulates APP processing, the findings of this thesis suggest that ABCA7 may have a role in maintaining cellular cholesterol homeostasis via cholesterol efflux to apoE acceptors and possibly by regulating intracellular trafficking of specific membrane proteins. In summary, ABCA7 was clearly shown in this thesis to have an effect on Aβ generation, clearance and degradation. Furthermore, ABCA7 expression was also affected by Aβ deposition in the brain. Even though more work is required to further elucidate the exact function of ABCA7, the findings presented in this thesis provide the first clues to the function of ABCA7 in the brain.

No description available.

Alzheimer's disease (AD) is the most common cause of a progressive decline of cognitive function in aged humans. It is thought to be a result of the formation of amyloid plaques in the brain that are largely composed of β-amyloid (Aβ), which is one of the cleavage products of the amyloid precursor protein (APP). The amyloidogenic processing of APP to produce the Aβ peptides requires sequential proteolytic cleavages by the β- and γ-secretases. APP is first cleaved by the β-secretase to produce APP-C99, and this product is a substrate for further processing by the γ-secretase that cleaves within its transmembrane domain to produce N-terminal Aβ peptides and the C-terminal APP intracellular domain (AICD). On the basis of similarities to the Notch processing pathway, it has been postulated that the AICD may play a role in gene regulation following its release in response to some form of extracellular signal. In order to better understand the production and fate of the AICD, I have investigated the potential for exploiting a cell-free system to study its generation and properties. Having generated a number of model APP-derived fragments and shown them to be efficiently membrane integrated in vitro, I went on to study AICD production. I discovered that AICD-like fragments are extremely labile when synthesised in a rabbit reticulocyte lysate system and are rapidly degraded via a metalloproteinase, most likely the insulin degrading enzyme (IDE). The in vitro stability of these model AICD-like fragments was dependent upon the precise chain length of the polypeptide and N-terminal processing may preface the activity of IDE in vitro. The rapid degradation of the AICD in vitro is in close agreement with previous in vivo studies, and taken together such data are consistent with a role for the AICD in a signalling pathway of some form. A variety of approaches were also taken to try to generate the AICD by the γ-secretase mediated cleavage of the APP-C99 fragment, a biologically relevant substrate. In no case was any evidence of such cleavage observed in vitro and hence I conclude that the endoplasmic reticulum does not possess an active form of the γ-secretase. Preliminary in vivo-based studies did provide evidence for the γ-secretase cleavage of APP-C99 fragments, consistent with current models implying that such processing takes place at the cell surface and/or in endosomes and not at the endoplasmic reticulum.

Mounting evidence indicates an involvement of inflammation in the pathogenesis of Alzheimer’s disease. While there are other mechanisms involved, it is this role of inflammatory processes that we wish to investigate. Triptolide is the major constituent in the Chinese herb, Tripterygium wilfordii Hook F, and has been used for centuries as part of Chinese herbal medicine. The four ringed structure has close homology to drugs of the steroid class and it has been shown to be beneficial as an anti-inflammatory for rheumatoid arthritis and for treatment of certain cancers. The aim of this study was to evaluate the potential therapeutic effect of Triptolide on the neuropathology and deficits of spatial 6 learning and memory in amyloid precursor protein (APP) and presenilin 1 (PS1) doubletransgenic mice, a well established Alzheimer’s disease (AD) mouse model. After treatment of APP/PS1 mice with Triptolide (40μg/kg, three times weekly,), initiated when the mice were 5 months old, for as little as 8 weeks, significant decrease in β-amyloid (Aβ) deposition and microglia activation was observed. Moreover, Triptolide treatment robustly rescued spatial memory deficits observed in APP/PS1 mice. However, APP processing, tau hyperphosphorylation, and the activities of the two major kinases involved in tau hyperphosphorylation, cyclin dependent kinase 5 (cdk5) and glycogen synthase kinase 3β (GSK3β) were not affected by the Triptolide treatment. Based on the recent finding for the inhibitory effect of Triptolide on Aβ-induced production of pro-inflammatory cytokines from microglia, we propose that Triptolide treatment may have beneficial properties in halting glial activation and help restore an immune system that fights plaque deposition. Although the exact mechanism of action has yet to be deduced, the increase in APP CTFs while having a significant decrease in amyloid plaque deposition suggests that alterations in gamma secretase activity may be a possible answer. Currently, these results support the use of Triptolide as an effective therapeutic to prevent the progression of Alzheimer’s disease.

<p>Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, neuropathologically characterized by neurofibrillay tangles and deposition of amyloid-β (Aβ) peptides. Several mutations in the gene for amyloid precursor protein (APP) cause familial AD and affect APP processing leading to increased levels of Aβ42. However, the Arctic Alzheimer mutation (APP E693G) reduces Aβ levels. Instead, the increased tendency of Arctic Aβ peptides to form Aβ protofibrils is thought to contribute to the pathogenesis. </p><p>In this thesis, the pathogenic mechanisms of the Arctic mutation were further investigated, specifically addressing if and how the mutation affects APP processing. Evidence of a shift towards β-secretase cleavage of Arctic APP was demonstrated. Arctic APP did not appear to be an inferior substrate for α-secretase, but the availability of Arctic APP for α-secretase cleavage was reduced, with diminished levels of cell surface APP in Arctic cells. Interestingly, administration of the fatty acid docosahexaenoic acid (DHA) stimulated α-secretase cleavage and partly reversed the effects of the Arctic mutation on APP processing.</p><p>In contrast to previous findings, the Arctic mutation generated enhanced total Aβ levels suggesting increased Aβ production. Importantly, this thesis illustrates and explains why measures of both Arctic and wild type Aβ levels are highly dependent upon the Aβ assay used, with enzyme-linked immunosorbent assay (ELISA) and Western blot generating different results. It was shown that these differences were due to inefficient detection of Aβ oligomers by ELISA leading to an underestimation of total Aβ levels. </p><p>In conclusion, the Arctic APP mutation leads to AD by multiple mechanisms. It facilitates protofibril formation, but it also alters trafficking and processing of APP which leads to increased steady state levels of total Aβ, in particular at intracellular locations. Importantly, these studies highlight mechanisms, other than enhanced production of Aβ peptide monomers, which could be implicated in sporadic AD.</p>

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, neuropathologically characterized by neurofibrillay tangles and deposition of amyloid-β (Aβ) peptides. Several mutations in the gene for amyloid precursor protein (APP) cause familial AD and affect APP processing leading to increased levels of Aβ42. However, the Arctic Alzheimer mutation (APP E693G) reduces Aβ levels. Instead, the increased tendency of Arctic Aβ peptides to form Aβ protofibrils is thought to contribute to the pathogenesis. In this thesis, the pathogenic mechanisms of the Arctic mutation were further investigated, specifically addressing if and how the mutation affects APP processing. Evidence of a shift towards β-secretase cleavage of Arctic APP was demonstrated. Arctic APP did not appear to be an inferior substrate for α-secretase, but the availability of Arctic APP for α-secretase cleavage was reduced, with diminished levels of cell surface APP in Arctic cells. Interestingly, administration of the fatty acid docosahexaenoic acid (DHA) stimulated α-secretase cleavage and partly reversed the effects of the Arctic mutation on APP processing. In contrast to previous findings, the Arctic mutation generated enhanced total Aβ levels suggesting increased Aβ production. Importantly, this thesis illustrates and explains why measures of both Arctic and wild type Aβ levels are highly dependent upon the Aβ assay used, with enzyme-linked immunosorbent assay (ELISA) and Western blot generating different results. It was shown that these differences were due to inefficient detection of Aβ oligomers by ELISA leading to an underestimation of total Aβ levels. In conclusion, the Arctic APP mutation leads to AD by multiple mechanisms. It facilitates protofibril formation, but it also alters trafficking and processing of APP which leads to increased steady state levels of total Aβ, in particular at intracellular locations. Importantly, these studies highlight mechanisms, other than enhanced production of Aβ peptide monomers, which could be implicated in sporadic AD.