Investigating the effects of the Alzheimer's disease-associated amyloid β-peptide on intracellular calcium homeostasis

• Main Author: Allan, Laura Elizabeth
• Published: University of Cambridge 2010
• Subjects: 612.8
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.595451

I investigated the effects of Aβ₄₂ on the Ca²⁺ signalling capacity of human neuroblastoma SH-SY5Y cells and primary hippocampal cultures. I developed an <i>in vitro</i> model system of dissociated hippocampal neurons and glial cells in order to reflect as closely as possible the mature hippocampus. Extensive characterisation of the culture revealed that functional neuronal networks were established by day <i>in vitro </i>11, as demonstrated by the occurrence of spontaneous oscillations in both membrane potential and intracellular Ca²⁺ levels. Neurons exhibited functional ionotropic and metabotropic signalling systems which, in turn, rendered them sensitive to cell death induced by excitotoxic stimuli. Samples of synthetic Aβ₄₂ were prepared according to two published protocols. One protocol produced Aβ₄₂ samples which exhibited highly dynamic aggregation kinetics, the other produced homogeneous Aβ₄₂ oligomers which were stable in their conformational state for up to 24 hours. While both Aβ₄₂ preparations impaired cell viability following 24-hour treatment, only Aβ₄₂ oligomers elicited robust Ca²⁺ responses following their extracellular application to cells. Aβ₄₂ oligomers elicited distinct but different effects on the Ca²⁺ signalling capacity of SH-SY5Y cells and primary hippocampal neurons, respectively. in SH-SY5Y cells, Aβ₄₂ oligomers acted to deplete the content of the intracellular ER Ca²⁺ store, in part through InsP₃ receptors and in part through an as yet unidentified leak pathway. In primary hippocampal neurons, the application of Aβ₄₂ oligomers resulted in a sustained and elevated increase in intracellular Ca²⁺ concentrations. It is postulated that these early cellular events, although distinct, will ultimately converge on a common pathway resulting in dysregulation of Ca²⁺ homeostasis and cell death. Thus, the findings of this thesis support the mounting body of evidence implicating the role of Ca²⁺ dysregulation in mediating the neurotoxic effects of Aβ₄₂ oligomers.