Engineering tools to analyze beta-amyloid's effect on cellular mechanisms linked to the neurodegeneration observed in Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that primarily affects the elderly population above 65 years of age. The disease is characterized by cognitive impairment, neuronal degeneration and eventual cell death. Current treatments for AD only slow down the progression of the disease and minimize its symptoms. Due to a lack of consensus of hypothesis among researchers, the cellular mechanism behind AD is not completely understood. Consequently, no effective treatment that stops or reverses the progression of the disease has been identified yet. In our work we applied analytical techniques and engineered tools to study the cellular mechanism of AD. We based our studies on the amyloid hypothesis, which states that beta-amyloid peptide, the primary component of senile plaques found in the brains of AD patients is the chief proponent of the disease. This hypothesis is supported in the past by research where Abeta in its aggregated form was found to be toxic to neuronal cells. We worked with the toxic forms of the peptide, Abeta 1-40 and 1-42 aggregated into fibrils, in all our studies. We first studied the interaction of Abeta with differentiated SY5Y neuroblastoma cells. We studied Abeta peptide's residue specific interactions in the vicinity of the integrin receptor on the cell membrane. We deduced from our results that Abeta's cellular interaction is influenced by both biological and electrostatic factors, and that certain residues of Abeta including its first seven residues affect the extent of interaction. These findings provide information for development of potential inhibitors of Abeta cellular binding. We next focused on signaling pathways related to the learning, memory and survival processes in SY5Y cells that are possibly perturbed by Abeta upon binding to the cell membrane. Using a combination of experimental studies and kinetic modeling, we investigated the effect of Abeta on the set of cellular signaling pathways and identified a plausible mechanism for its toxicity. This toxicity effect of Abeta was attenuated upon blocking specific pathways in the mechanism under the experimental conditions conducted. These findings provide insight for possible therapeutics that inhibit Abeta's toxic activity . While binding of Abeta to cells and the subsequent downstream signaling are important intial steps in tha AD pathway, these phenomenon cannot explain long term manifestations of the disease. Recent evidence points to the role of epigenetics in AD, and proposes DNA methylation to be a symptom of Abeta's effect on cells. As such, we began to explore the effect of Abeta on epigenetics of cells, specifically methylation changes in SY5Y cells and neural stem cells (NSCs). We first attempted to develop a novel technique for detection of methylation changes in a target DNA sequence. The method was partly successful in that target DNA was captured and its methylation levels detected, but the level of detection was lower than expected. The results from our technique development provide a basis for a cost-effective assay of methylation changes in cellular DNA samples. We continued our work on study of methylation changes upon Abeta exposure using established microarray technology.We examined global DNA and gene sequence specific methylation changes in SY5Y cells as well as NSCs upon exposure to Abeta. We accomplished this through a genome wide microarray analysis of methylation changes in cells, focusing especially on gene regions implicated in AD and involved in the regulation of the differentiation state of NSCs. The findings from these studies provide insights into Abeta's effect on the methylation levels of cells that can be linked to its effect on cellular differentiation and perturbation of events in the progression of AD. This work elucidates the impact of epigenetics on AD and could lead to the development of effective stem cell therapies for AD. In summary, we studied the Abeta peptide's interaction with the cellular membrane, the subsequent perturbation of intracellular signaling pathways and the effect on the epigenetic makeup of cells. These studies provide a picture of Abeta's effects on cellular processes related to their learning, memory, differentiation and survival that could have an impact on AD. The results pinpoint us and other researchers towards future treaments for this disease

Engineering tools to analyze beta-amyloid's effect on cellular mechanisms linked to the neurodegeneration observed in Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that primarily affects the elderly population above 65 years of age. The disease is characterized by cognitive impairment, neuronal degeneration and eventual cell death. Current treatments for AD only slow down the progression of the disease and minimize its symptoms. Due to a lack of consensus of hypothesis among researchers, the cellular mechanism behind AD is not completely understood. Consequently, no effective treatment that stops or reverses the progression of the disease has been identified yet. In our work we applied analytical techniques and engineered tools to study the cellular mechanism of AD. We based our studies on the amyloid hypothesis, which states that beta-amyloid peptide, the primary component of senile plaques found in the brains of AD patients is the chief proponent of the disease. This hypothesis is supported in the past by research where Abeta in its aggregated form was found to be toxic to neuronal cells. We worked with the toxic forms of the peptide, Abeta 1-40 and 1-42 aggregated into fibrils, in all our studies. We first studied the interaction of Abeta with differentiated SY5Y neuroblastoma cells. We studied Abeta peptide's residue specific interactions in the vicinity of the integrin receptor on the cell membrane. We deduced from our results that Abeta's cellular interaction is influenced by both biological and electrostatic factors, and that certain residues of Abeta including its first seven residues affect the extent of interaction. These findings provide information for development of potential inhibitors of Abeta cellular binding. We next focused on signaling pathways related to the learning, memory and survival processes in SY5Y cells that are possibly perturbed by Abeta upon binding to the cell membrane. Using a combination of experimental studies and kinetic modeling, we investigated the effect of Abeta on the set of cellular signaling pathways and identified a plausible mechanism for its toxicity. This toxicity effect of Abeta was attenuated upon blocking specific pathways in the mechanism under the experimental conditions conducted. These findings provide insight for possible therapeutics that inhibit Abeta's toxic activity . While binding of Abeta to cells and the subsequent downstream signaling are important intial steps in tha AD pathway, these phenomenon cannot explain long term manifestations of the disease. Recent evidence points to the role of epigenetics in AD, and proposes DNA methylation to be a symptom of Abeta's effect on cells. As such, we began to explore the effect of Abeta on epigenetics of cells, specifically methylation changes in SY5Y cells and neural stem cells (NSCs). We first attempted to develop a novel technique for detection of methylation changes in a target DNA sequence. The method was partly successful in that target DNA was captured and its methylation levels detected, but the level of detection was lower than expected. The results from our technique development provide a basis for a cost-effective assay of methylation changes in cellular DNA samples. We continued our work on study of methylation changes upon Abeta exposure using established microarray technology.We examined global DNA and gene sequence specific methylation changes in SY5Y cells as well as NSCs upon exposure to Abeta. We accomplished this through a genome wide microarray analysis of methylation changes in cells, focusing especially on gene regions implicated in AD and involved in the regulation of the differentiation state of NSCs. The findings from these studies provide insights into Abeta's effect on the methylation levels of cells that can be linked to its effect on cellular differentiation and perturbation of events in the progression of AD. This work elucidates the impact of epigenetics on AD and could lead to the development of effective stem cell therapies for AD. In summary, we studied the Abeta peptide's interaction with the cellular membrane, the subsequent perturbation of intracellular signaling pathways and the effect on the epigenetic makeup of cells. These studies provide a picture of Abeta's effects on cellular processes related to their learning, memory, differentiation and survival that could have an impact on AD. The results pinpoint us and other researchers towards future treaments for this disease

Role of N-terminal residues in Aβ interactions with integrin receptor and cell surface

Abstractbeta-Amyloid (Aβ) is the primary protein component of senile plaques in Alzheimer's disease (AD) and is believed to play a role in its pathology. To date, the mechanism of action of Aβ in AD is unclear. We and others have observed that Aβ interacts either with or in the vicinity of the α6 sub-unit of integrin, and believe this may be important in its interaction with neuronal cells. In this study, we used confocal microscopy and flow cytometry to explore the residue specific interactions of Aβ40 with the cell surface and the α6 integrin receptor sub-unit. We probed the importance of the RHD sequence in Aβ40 and found that removal of the residues or their mutation using the Aβ8-40 or the D7N early onset AD sequence, respectively, led to a greater interaction between Aβ40 and an antibody bound to the α6-integrin sub-unit, as measured by fluorescence resonance energy transfer (FRET). These results suggest that the RHD sequence of Aβ40 does not mediate Aβ–α6 integrin interactions. However, the cyclic RGD mimicking peptide, Cilengitide, reduced the measured interaction between Aβ40 fibrils without the RHD sequence and an antibody bound to the α6-integrin sub-unit. We further probed the role of electrostatic forces on Aβ40–cell interactions and observed that the Aβ sequence that included the N-terminal segment of the peptide had reduced cellular binding at low salt concentrations, suggesting that its first 7 residues contribute to an electrostatic repulsion for the cell surface. These findings contribute to our understanding of Aβ–cell surface interactions and may provide insight into development of novel strategies to block Aβ–cell interactions that contribute to pathology in Alzheimer's disease

A Critical Role for HlgA in Staphylococcus aureus Pathogenesis Revealed by A Switch in the SaeRS Two-Component Regulatory System

Cytolytic pore-forming toxins including alpha hemolysin (Hla) and bicomponent leukotoxins play an important role in the pathogenesis of Staphylococcus aureus. These toxins kill the polymorphonuclear phagocytes (PMNs), disrupt epithelial and endothelial barriers, and lyse erythrocytes to provide iron for bacterial growth. The expression of these toxins is regulated by the two-component sensing systems Sae and Agr. Here, we report that a point mutation (L18P) in SaeS, the histidine kinase sensor of the Sae system, renders the S. aureus Newman hemolytic activity fully independent of Hla and drastically increases the PMN lytic activity. Furthermore, this Hla-independent activity, unlike Hla itself, can lyse human erythrocytes. The Hla-independent activity towards human erythrocytes was also evident in USA300, however, under strict agr control. Gene knockout studies revealed that this Hla-independent Sae-regulated activity was entirely dependent on gamma hemolysin A subunit (HlgA). In contrast, hemolytic activity of Newman towards human erythrocytes from HlgAB resistant donors was completely dependent on agr. The culture supernatant from Newman S. aureus could be neutralized by antisera against two vaccine candidates based on LukS and LukF subunits of Panton-Valentine leukocidin but not by an anti-Hla neutralizing antibody. These findings display the complex involvement of Sae and Agr systems in regulating the virulence of S. aureus and have important implications for vaccine and immunotherapeutics development for S. aureus disease in humans