It is widely accepted that A-beta (Aβ) generated from amyloid precursor protein (APP) oligomerizes and fibrillizes to form neuritic plaques in the Alzheimer’s disease (AD) brain, yet little is known about the contribution of APP preceding AD pathogenesis. Our data presented here suggest that APP has a functional role in cell cycle regulation and proliferation. First, we demonstrat that APP is pathologically phosphorylated at Thr668 and that P-APP localizes to the centrosomes. Furthermore, P-APP is proteolytically processed in a cell cycle -dependent manner to generate its pathogenic metabolites. Using Stable Isotope Labeling by Amino Acids in Culture (SILAC) and mass spectrometry analyses, we also show that expression of APP results in the expression of proliferation-associated proteins and the phosphorylation of proteins associated with cell cycle regulation and transcription. Here, we demonstrate that APP expression and oligomeric Aβ42 elicit Ras/ERK signaling cascade and glycogen synthase kinase3 (GSK3) activation. Both ERK and GSK3 are known to induce hyperphosphorylation of tau and of APP at Thr668, and our findings suggest that aberrant signaling by APP facilitates these events. Supporting this notion, analysis of human brain samples show increased expression of Ras, activation of GSK3 and phosphorylation of APP and tau, which correlate with Aβ levels in the AD brains. Furthermore, treatment of primary rat neurons with Aβ recapitulate these events and show enhanced Ras-ERK signaling, GSK3 activation, upregulation of cyclin D1, and phosphorylation of APP and tau. The finding that Aβ induces Thr668 phosphorylation on APP, which we show enhances APP proteolysis and Aβ generation, denotes a vicious feed-forward mechanism by which APP and Aβ promote tau hyperphosphorylation and neurodegeneration in AD. Based on these results we hypothesize that aberrant proliferative signaling by APP plays a fundamental role in AD neurodegeneration and an inhibition of this would impede the mitotic catastrophe and neurodegeneration observed in AD.

Magister Pharmaceuticae - MPharm / The cascade of neurotoxic events involved in the pathogenesis of Alzheimer's disease may explain the inefficacy of currently available treatment based on acetylcholinesterase inhibitors (AChEI - donepezil, galantamine, rivastigmine) and N-methyl-D-aspartate (NMDA) antagonists (memantine). These drugs were designed based on the "one-moleculeone- target" paradigm and only address a single target. Conversely, the multi-target drug design strategy increasingly gains recognition. Based on the versatile biological activities of tacrine, trolox and β-carboline derivatives, the attention they have received as lead structures for the design of multifunctional drugs for the treatment of Alzheimer's disease, and the topology of the active site of AChE, we have designed tacrine-trolox and tacrine-tryptoline hybrids with various linker chain lengths. The aim with these hybrids was to provide additive or synergistic therapeutic effects that might help overcome the limitation of current anti Alzheimer's disease drugs. All synthesized compounds were designed from lead structures (tacrine, tryptoline and trolox) to obtain cholinesterase (ChE) multisite binders and multifunctional AD agents. The study was rationalized by docking all structures in the active site of TcAChE using Molecular Operating Environment (MOE) software before proceeding with the synthesis. ChE inhibition was assessed in a UV enzyme inhibition assay using Ellman's method. Antioxidant activities were assessed using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH.) absorbance assay. The hybrids containing the trolox moiety (compounds 8a-e) showed moderate to high AChE inhibitory activity in the nano to micro molar range (IC₅₀: 17.37 - 2200 nM), BuChE inhibition was observed in the same range (IC₅₀: 3.16 – 128.82 nM), and free radical scavenging activities in micro molar range (IC50: 11.48 – 49.23 µM). These are comparable or slightly higher than their reference compounds donepezil (AChE IC₅₀ = 220 nM), tacrine (BuChE IC₅₀: 14.12 nM), and trolox (DPPH IC₅₀: 17.57 µM). The hybrids with longer linker chain lengths, 6 and 8 carbons (8d and 8e), showed better ChE inhibitory activity than the shorter ones, 2, 3, and 4 carbons (8a-c respectively). This correlates well with literature. Free radical scavenging activities, however, seems not to be significantly affected by varying linker chain lengths. The hybrid compound (14) containing the tryptoline moiety linked with a 7 carbon spacer displayed the best AChE and BuChE inhibitory activity (IC₅₀ = 17.37 and 3.16 nM) but poor free radical scavenging activity. Novel anti-Alzheimer's disease drugs with multi-target neuroprotective activities were thus obtained and hybrid molecules that exhibit good ChE inhibition (8d, 8e and 14) and anti-oxidant (8d and 8e) activity were identified as suitable candidates for further investigation. / National Research Foundation (NRF)

No description available.

Light chain amyloidosis (AL) is associated with high mortality, especially in patients with advanced cardiovascular involvement. It is caused by toxicity of misfolded light chain proteins (LC) in vascular, cardiac, and other tissues. There is no treatment to reverse LC tissue toxicity. We tested the hypothesis that nanoliposomes composed of monosialoganglioside, phosphatidylcholine, and cholesterol (GM1 ganglioside-containing nanoliposomes [NLGM1]) can protect against LC-induced human microvascular dysfunction and assess mechanisms behind the protective effect.

Alzheimer's disease (AD) is a worldwide, incurable disease, and the most common form of dementia. Numbers of cases are rising, and since its discovery the only approved medications have treated only the symptoms, not the pathological cause. With the cost to society rising, the debilitating nature of the disease and the pressure put on the family members and support network of patients, disease modifying therapies are in dire need. Current models have proven an invaluable tool with which to study certain aspects of the disease and the genetics behind it, however the lack of clinically approved medications in the last 20 years suggests new models are needed. Based on the amyloid cascade hypothesis, this thesis initially characterises two models of β-Amyloid oligomer (Aβo) induced cognitive deficits. Both models are created by ICV injection of soluble Aβo into the brain of rat. The models differ only by the molecular weight of the Aβo 1-42, one, referred to as low molecular weight (LMW) Aβo, with stable dimers, trimers and tetramers, the other, referred to as high molecular weight (HMW) Aβo, consisting of assemblies ranging from ~50 to ~150 kDa. It was found that behavioural deficits were similar between the two, with a robust object recognition deficit, but no working memory deficit. Both models also showed a deficit in the synaptic marker PSD-95; however the LMW Aβo caused a deficit in the frontal cortex, whereas the HMW Aβo caused a hippocampal deficit. The role of the cellular prion protein (PrPC) was explored, by blocking its binding to Aβo with the antibody 6D11. Interestingly the two models showed different results. The HMW Aβo deficits were completely blocked by the 6D11 application, however the LMW Aβo deficits were only partially prevented. Finally, Fasudil, a vasodilator approved in parts of Asia, was used to inhibit Rho-kinase, showing a prevention of the cognitive deficits in the HMW Aβo model. The results of this thesis show the ICV administration of Aβo to be a useful model for investigating the effects of Aβo, provides a platform with which to study the differing effects of Aβo with different oligomeric assemblies, and a model to test therapeutic strategies with relevance to AD.

"Alzheimer’s disease (AD) is the most widespread neurodegenerative disorder, affecting approximately 20 million people worldwide. AD pathology is primarily characterized by the formation of extracellular amyloid plaques resulting from the aggregation of insoluble amyloid-beta 1-42 (A-beta), and neurofibrillary tangles (NFT’s) resulting from intracellular aggregation of hyperphosphorylated tau protein. The current FDA-approved AD treatments do not stop or reverse neurodegeneration, but only treat the symptoms by increasing acetylcholine neurotransmitter. Our laboratory is attempting to provide an additional therapeutic approach by using neurotrophic factors to block apoptosis or to restore neurons. We previously demonstrated that, in an in vitro model for AD, hEPN-1 neurotrophic factor mimetics can block synthetic A-beta-induced neuronal cell death when added to cultures, presumably by blocking caspase activation. In this thesis, we extended these findings to study the effect of A-beta and hEPN-1 on tau hyperphosphorylation (as measured by immunoblots with phospho-specific antibodies) and nuclear DNA fragmentation (as measured by TUNEL staining), both in vitro and in vivo in AD transgenic mice. We found that A-beta induces the hyperphosphorylation of tau in both mouse N2a and human SHSY neuronal cells, and that hEPN-1 may lower this phosphorylation in N2a cells. Furthermore, we discovered that hEPN-1 can reduce nuclear DNA fragmentation when added both simultaneously to A-beta and 3 and 6 hours post A-beta addition. Finally, in vivo hEPN-1 may lower both tau hyperphosphorylation and caspase-7 related protein (C7RP) in AD transgenic (Tg) mice. The overall results validate our in vitro AD model, show the efficacy of hEPN-1 at blocking A-beta-induced DNA fragmentation even when added post-insult, and show that hEPN-1 may work in an AD mouse model. However, more studies must be conducted to confirm these findings. "

We study the conformational change of insulin fibril growth from three aspects: the impact of (i) incubation time; (ⅱ) nano-particles; (iii) and ion added. We used circular dichroism (CD) spectroscopy and fourier transform infrared spectroscopy (FT-IR) to obtain the structural transition of the insulin, and gain the morphology information of fibril by atomic force microscopy (AFM) and transmission electron microscopy (TEM). We show that the insulin transform from £\-helix to £]-sheet structure as increased incubated time. The addition of Au nanoparticles (NPs) caused the formation of coordination bond with insulin fiber and produced shorter and thicker insulin fibril . The Fe3O4 NPs, on the other hand, offered only van der Waals interaction toward insulin fibril. Hence they could be used to separate insulin fibril from solution. Finally, addition of salts can induce the conformation changes of insulin fibril ten times faster than that without salts. And the insulin fibril fragment was two or three times shorter than that produced without salts. At high salt concentration, insulin formed amorphous aggregates. This phenomenon was attribute to anions from salt: covering the surface charge of insulin fibril, they weaken the original electrostatic repulsion among insulin fibrils and result in their aggregation.

In this report, electrostatic force microscopy (EFM), zeta-potential analyzer, circular dichroism Spectrophotometer and Fourier transform infrared spectroscopy are used to study the electrostatic property of surface on insulin fibril. EFM provides a simultaneous probe of topography and electrostatic property with highly local resolution in nanometer-scale. To understand the correlation between charge distribution on fibril surface and structure transformation, we controlled the incubation time and salt concentration. The results show that the surface charge increases with incubation time, and with the salt concentration increased, the pitch is found to decrease, variation of charge distribution are increased. In addition, we also have evaluated influence of amino acid sequence on growth rate of fibril associated with bovine, porcine and human insulin by atomic force microscopy and circular dichroism Spectrophotometer. The experimental results show that the number of hydrophilic groups on the peptide sequence will affect the speed of fibril generated, and the location of hydrophilic groups on the peptide sequence will affect the stability of the fibril under the frozen environment.

No description available.

The inverse scattering problem is based on the scattering theory in physics, where measured data such as radiation from an object is used to determine the unique structure of the object in question. This approach has been widely successful in fields ranging from geophysics and medical imaging, to quantum field theory. In 1996 Henrik Flyvbjerg suggested that a similar approach could be used to study a reaction far from equilibrium of the self-assembly of a nucleation dependent biopolymer and, under certain conditions, uniquely determine the kinetics of the assembly. Here we use this approach to elucidate the unique structure of human islet amyloid polypeptide, also known as amylin, in-vitro. We use a systematic phenomenological analysis of the amount of monomer in fibril, of amylin, for various initial concentrations from an unstructured monomer pool. Using the assumption that nucleation is the rate-limiting step in fibril formation, we invoke mass action to develop our model. We find that the fibrillogenesis of amylin is well described by a nucleation dependent polymerization event that is characteristic of the sigmoidal shape of the reaction profile generated by our data. Furthermore, we find a second nucleation event is needed to accurately match model predictions to the observed data for the kinetic profiles of fibril formation, and the experimental length distributions of mature fibrils from in-vitro assays. This analysis allows for the theoretical determination of each step of assembly in the nucleation process. Specifically, we find the number of steps to nucleation, the size of each oligomer formed in the nucleation process, the nucleus size, and the elongation kinetics of fibrils. The secondary nucleation process is found to be a fibril dependent surface mediated nucleation event and is similar in reaction order to the primary nucleation step. Model predictions are found to be congruent with experimental assay results of oligomer populations and monomer concentration. We demonstrate that, a persistent oligomer formation is a natural and necessary consequence of nucleated fibril formation, given certain qualitative features of the kinetic profile of fibril formation. Furthermore, the modeling assumptions about monomer and fibril mass are in agreement with experiment.