Engineering tools to analyze beta-amyloid's effect on cellular mechanisms linked to the  neurodegeneration observed in Alzheimer's disease
Creators: name: Venkatasubramaniam, Arundhathi
Publisher: name: Maryland Shared Open Access Repository
Year: 2014
Subjects: Alzheimer
amyloid
epigenetics
FRET
integrin
signaling

description: 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.