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Amylin is a 37 amino acid long peptide that aggregates into toxic oligomers and fibrils. Since amylin is secreted by and also acts on pancreatic beta cells, type II diabetes is classified as an amyloidogenic disease. This study focuses on the development of a cost effective chemical synthetic strategy for amylin synthesis as previous studies relied on extremely expensive pseudoproline derivatives. Furthermore, commercially available amylin varies between batches and also contains impurities that could generate anomalies and affect reproducibility of experiments. Secondly, chemically synthesized non-methylated and N-methylated derivatives of amylin were shown to inhibit toxicity of full length amylin. A fluorescentlylabeled chemically synthesized derivative of amylin was used to track cellular localization of amylin via confocal microscopy. Amylin aggregation kinetics was established using a surface plasmon resonance (SPR) biosensor. In addition, nanoparticle tracking analysis (NTA) was used as a novel technique to determine the size of oligomers over real time. This technology indicated that the size range of the toxic species of amylin is between 200-300 nm. Furthermore, it can be suggested that NTA could potentially be developed into a screening tool for inhibitors of amylin-mediated cytotoxicity. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2012.

Despite being first identified over a century ago, Alzheimer’s disease (AD) is a complex neurological disorder that still has not been properly characterized. Most cases are sporadic in nature, with an unidentifiable cause, but early-onset familial Alzheimer’s disease (FAD) is induced by genetic mutations in certain key genes. FAD mutations in the full length amyloid precursor protein (flAPP) increases production of the amyloid beta (Aβ) peptide responsible for plaque formation commonly associated with the disease, leading to neuronal death. A mutation in the PS1 gene (mPS1) results in increased APP cleavage into Aβ1-42, also leading to early AD formation. Although discoveries of FAD mutations have enabled concentrated studies into AD pathogenesis, its cause is still unknown. In this thesis, experimental projects were designed to study how signaling pathways associated with markers of AD, including APP and PS1 gene mutations, could result in neuronal dysfunction associated with disease pathology, and how these pathways could be manipulated for use as potential therapeutic targets. Cortical neurons isolated from FAD mPS1 mice (expressing the Met146Val mPS1 protein) were analyzed to establish neuronal viability in response to Aβ1-42 insult compared to healthy neurons. mPS1 neurons were no more susceptible to cell death compared to wild-type neurons, because of an increased activation of the transcription factor nuclear factor kappa B (NF-κB) protein brought about by elevated endoplasmic reticulum (ER) calcium release due to the PS1 mutation. However, NF-κB inhibition in the mPS1 neurons caused increased pro-apoptotic protein CHOP expression leading to significantly higher cell death versus controls when neurons were exposed to Aβ1-42. Following this study, the role of the neurotrophic protein neuregulin on cytoplasmic calcium levels of hippocampal neurons was examined, with the intent of assessing the contributioin of that signaling pathway to AD neuropathology in AD transgenic mice. Neuregulin has been shown to modify glutamatergic channels at neuronal synapses, but how this could affect cytoplasmic calcium levels in neurons was uncertain. Long term treatment (24 hours), but not short-term (1 hr), with neuregulin increased glutamatergic-induced intracellular calcium levels in hippocampal neurons, through a PI3K-mediated mechanism. This study demonstrated that inhibition of the NRG/ErbB axis could be a possible therapeutic target to reduce excitotoxic levels of calcium leading to neuronal death in AD, or enhance synaptic plasticity and memory in AD-initiated areas of deficit. Finally, interactions between the neurotrophic insulin pathway and amyloid peptides were studied using an amyloid precursor protein (APP) overexpressing mouse model, the TgCRND8 strain. Despite insulin depletion induced by streptozotocin injection, young diabetic TgCRND8 mice displayed no impairment in insulin signaling compared to controls, likely due to activation of the insulin signaling pathway by sAPPα. This indicates a possible biological role for sAPPα that prevents diabetic-induced insulin signaling impairment. Thus, the data from these three projects elucidated different components of AD pathogenesis and possibly targets of future AD treatment.

Neuroinflammation occurs in Alzheimer’s disease (AD) brain, and plays a role in neurodegeneration. The main aim of this study was to determine how treatments with exogenous apolipoprotein E (ApoE2, E3 and E4 isoforms), a genetic risk factor for AD, affects the amyloid-β (Aβ) induced inflammatory response in vitro in astrocytes. Recombinant, lipid-free ApoE4 was found not to affect Aβ-induced inflammation in rat astrocytes, while ApoE2 showed a protective effect. Mouse cells expressing human ApoE isoforms, which have similar lipidation and modification to native human ApoE, showed ApoE4 promoting inflammation, and no ApoE2 protective effect upon Aβ treatment. A Protein/DNA array was used to screen 345 transcription factors in rat astrocytes treated with Aβ and/or ApoE isoforms, in order to determine which contribute to the observed ApoE2 protection. Some candidates were validated by Western Blot or EMSA and/or by inhibition or activation. The findings suggest ApoE isoforms differentially regulate Aβ-induced inflammation, and multiple signalling pathways are involved in the process.

Despite being first identified over a century ago, Alzheimer’s disease (AD) is a complex neurological disorder that still has not been properly characterized. Most cases are sporadic in nature, with an unidentifiable cause, but early-onset familial Alzheimer’s disease (FAD) is induced by genetic mutations in certain key genes. FAD mutations in the full length amyloid precursor protein (flAPP) increases production of the amyloid beta (Aβ) peptide responsible for plaque formation commonly associated with the disease, leading to neuronal death. A mutation in the PS1 gene (mPS1) results in increased APP cleavage into Aβ1-42, also leading to early AD formation. Although discoveries of FAD mutations have enabled concentrated studies into AD pathogenesis, its cause is still unknown. In this thesis, experimental projects were designed to study how signaling pathways associated with markers of AD, including APP and PS1 gene mutations, could result in neuronal dysfunction associated with disease pathology, and how these pathways could be manipulated for use as potential therapeutic targets. Cortical neurons isolated from FAD mPS1 mice (expressing the Met146Val mPS1 protein) were analyzed to establish neuronal viability in response to Aβ1-42 insult compared to healthy neurons. mPS1 neurons were no more susceptible to cell death compared to wild-type neurons, because of an increased activation of the transcription factor nuclear factor kappa B (NF-κB) protein brought about by elevated endoplasmic reticulum (ER) calcium release due to the PS1 mutation. However, NF-κB inhibition in the mPS1 neurons caused increased pro-apoptotic protein CHOP expression leading to significantly higher cell death versus controls when neurons were exposed to Aβ1-42. Following this study, the role of the neurotrophic protein neuregulin on cytoplasmic calcium levels of hippocampal neurons was examined, with the intent of assessing the contributioin of that signaling pathway to AD neuropathology in AD transgenic mice. Neuregulin has been shown to modify glutamatergic channels at neuronal synapses, but how this could affect cytoplasmic calcium levels in neurons was uncertain. Long term treatment (24 hours), but not short-term (1 hr), with neuregulin increased glutamatergic-induced intracellular calcium levels in hippocampal neurons, through a PI3K-mediated mechanism. This study demonstrated that inhibition of the NRG/ErbB axis could be a possible therapeutic target to reduce excitotoxic levels of calcium leading to neuronal death in AD, or enhance synaptic plasticity and memory in AD-initiated areas of deficit. Finally, interactions between the neurotrophic insulin pathway and amyloid peptides were studied using an amyloid precursor protein (APP) overexpressing mouse model, the TgCRND8 strain. Despite insulin depletion induced by streptozotocin injection, young diabetic TgCRND8 mice displayed no impairment in insulin signaling compared to controls, likely due to activation of the insulin signaling pathway by sAPPα. This indicates a possible biological role for sAPPα that prevents diabetic-induced insulin signaling impairment. Thus, the data from these three projects elucidated different components of AD pathogenesis and possibly targets of future AD treatment.

Alzheimer’s disease (AD), a progressive age-related neurodegenerative disorder is characterized by impairments in memory and cognitive functions. The two main pathogenic hallmarks associated with the progression of this multifactorial disease include accumulation of amyloid-beta (Aβ) plaques and the deterioration of the cholinergic system in the brain. Using cost-effective synthetic procedures, mono-, di-, and tri- substituted sym-triazine derivatives incorporating acetylcholine substrate analogues and aromatic phenyl moieties were synthesized for the targeted modulation of Aβ aggregation and acetylcholinesterase (AChE) activity. A subset of these sym-triazines demonstrated dual inhibition of Aβ fibrillization and AChE hydrolytic activity in vitro studies. These highly effective compounds were also shown to be well tolerated by differentiated human neuronal cells in cell viability tests. These novel compounds have the potential to undergo future in vivo pharmaceutical analysis and have a positive impact on the quality of life of the people living with this devastating disease and their caretakers.

Alzheimer’s disease (AD), a progressive age-related neurodegenerative disorder is characterized by impairments in memory and cognitive functions. The two main pathogenic hallmarks associated with the progression of this multifactorial disease include accumulation of amyloid-beta (Aβ) plaques and the deterioration of the cholinergic system in the brain. Using cost-effective synthetic procedures, mono-, di-, and tri- substituted sym-triazine derivatives incorporating acetylcholine substrate analogues and aromatic phenyl moieties were synthesized for the targeted modulation of Aβ aggregation and acetylcholinesterase (AChE) activity. A subset of these sym-triazines demonstrated dual inhibition of Aβ fibrillization and AChE hydrolytic activity in vitro studies. These highly effective compounds were also shown to be well tolerated by differentiated human neuronal cells in cell viability tests. These novel compounds have the potential to undergo future in vivo pharmaceutical analysis and have a positive impact on the quality of life of the people living with this devastating disease and their caretakers.

Abstract Alzheimer’s disease (AD) is a major public health concern worldwide, with an increasing prevalence in the elderly population. AD is a progressive neurological disorder of multi-faceted origin, where factors such as genetic mutations, biochemical changes, along with inflammatory cascade and soluble beta amyloid (Aβ) peptide, are thought to play a pivotal role in synaptic failure and neuronal death, ultimately leading to cognitive and neuropsychiatric decline in patients suffering from the disease. At present, there is no long-term cure for the disease, although there is access to pharmacotherapy that might improve cognitive and neuropsychiatric symptoms early in the course of the disease. The current pharmacological therapy for AD only provides symptomatic relief for a very short period of time. It is therefore of utmost importance to discover other pharmacological strategies that might delay the development of AD and slow down the disease progression in terms of cognitive decline and neurodegeneration. Elucidating the pathogenic mechanisms involved in AD neuropathogenesis is a major goal to find efficacious disease-modifying treatments. What remains to be understood completely are the intracellular pathways affected by Aβ protein which may lead to neurodegeneration in AD. Since phosphorylation and dephosphorylation mechanisms are crucial in the β-amyloid precursor protein (APP) metabolism, protein kinase C has emerged as one of the key regulators of the APP metabolism. Indeed, dysregulation of the PKC pathway might play a role in the intracellular mechanisms of neurodegeneration, but their effective involvement still remains elusive. Therefore, a detailed analysis of PKC pathways in established models of AD neurodegeneration is necessary and will form part of this work. Fucoidin is a sulphated polysaccharide extracted from edible brown seaweed, which has been shown to exhibit anti-inflammatory and anti-oxidant effects as well as being a neuroprotectant in various inflammatory diseases including hypoxic ischemia, atherosclerosis and Heyman nephritis. Therefore, fucoidin may have an inhibitory effect on the inflammatory mechanisms of AD. Little is known, however, about the effect of fucoidin on AD. Animal models of AD are extremely valuable for the discovery and development of new treatments. Rodents have been one of the preferred models for pharmacological and behavioural studies in AD. In this thesis, first aim was to establish a non-transgenic Aβ-induced AD model in rats. AD was induced utilising a published protocol which involved the bilateral injection of aggregated Aβ (1-42) into the CA3 subfield of the hippocampus in rat brain. Behavioural assessment with well defined tools such as the Morris water maze and T-maze were utilised to assess the impairment in spatial working memory in rats. Behavioural impairments along with increased astrocytosis and microgliosis were observed in this particular Aβ-induced AD model. In the established disease model, fucoidin (50 mg/kg/day and 25 mg/kg/day) and ibuprofen (50 mg/kg/day) were shown to provide a partial protective effect on impairment in memory function in the MWM behavioural task in rats treated prior to disease initiation and throughout the course of the study. In addition, the histopathological and quantitative analysis of AD brain sections showed a marked reduction in reactive glial fibrillary acidic protein (GFAP) and microglia in fucoidin (low and high dose) and ibuprofen treated Aβ injected rats compared to untreated Aβ injected rats. These results indicate that fucoidin may serve as a possible effective therapeutic approach to improve AD symptoms. There is strong evidence that PKC α and ε signalling pathways regulate important molecular events in memory impairment and neurodegenerative pathophysiology in AD. A possible neuroprotective mechanism of fucoidin involving attenuation of an Aβ-induced decrease in PKC ε phosphorylation using cultured SHSY5Y neuroblastoma cells as a model system was examined. Co-administration of fucoidin (2μM and 5 μM) with Aβ (1μM) abolished the inhibitory effect of Aβ on the phosphorylation of PKCε in a concentration-dependent manner as revealed by western blot analysis. These findings suggest that a possible mechanism underpinning the neuroprotective effect of fucoidin may be through prevention of A-induced inhibition of PKC phosphorylation and may serve as a possible therapeutic approach to improve AD symptoms. As cellular events that involve PKC are affected by Aβ in in vitro systems, it was necessary to examine whether PKC activity is also modulated by the Aβ treatment in vivo in our Aβ-peptide induced AD model. Therefore, the next aim was to assess the potential for fucoidin use as an intervention therapy in an established disease stage in the Aβ-peptide induced AD model. Intervention with fucoidin (50 mg/kg/day, i.p.) in the established disease stage partially prevented Aβ (1-42) mediated damage with respect to memory impairment, neuroinflammation and PKC ε phosphorylation in the in vivo AD model consistent with the in vitro findings in SHSY5Y cells.

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.

Appendum pasted into front end-papers. / Bibliography: leaves xiii-xliii. / xi, 195, xliii leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Traumatic brain injury (TBI) effects neuronal cell bodies (NCBs), axons and dendrites in a complex fashion, producing a spectrum of damage dependent on the initial injury and secondary effects. Accumulation of amyloid precursor (APP) in NSBs and axons is a feature of TBI. This accumulation may be due to impairment of the axonal transport of APP and/or upregulation of APP mRNA synthesis. This thesis hypothesizes that mechanical deformation, which is not severe enough to cause immediate cell death, results in increased APP mRNA and antigen expression as an acute phase response to injury. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 1999