Electrophysiological analysis of amyloid $\beta$-peptide toxicity in neuroblastoma cells {\it in vitro\/}

Previous studies have shown that amyloid $\beta$-peptide (A$\beta$P), a normally secreted protein found in low levels in different tissues, may play a role in the neurodegenerative changes seen in Alzheimer\u27s disease (AD) by disrupting calcium homeostasis in brain cells. Currently little is known regarding the cellular mechanisms that underlay the effects of A$\beta$P. The objective of this thesis was to examine possible ionic mechanisms involved in A$\beta$P-induced neurodegeneration. The hypothesis of these studies was that A$\beta$P alters calcium flux by modifying endogenous calcium channel activity and/or by forming calcium-permeable channels de novo in the neuronal membrane. In these studies, patch clamp electrophysiological recording technique was used to monitor A$\beta$P-induced changes in whole-cell and single-channel cation currents in the N1E-115 neuroblastoma (NB) cell and synthetic vesicle membrane.^ In whole-cell experiments, incubation of differentiated N1E-115 NB cells with toxic levels of a 40 amino acid A$\beta$P fragment (A$\beta$P1-40) increased the peak amplitude of the nimodipine-sensitive calcium current and shifted the activation and peak-current potential towards more positive voltages. Likewise, in single-channel studies, incubation with A$\beta$P increased both open probability (P$\sb{\rm open})$ and voltage at half-maximum P$\sb{\rm open}$ for calcium channels. By contrast, the highly toxic A$\beta$P25-35 and human amylin, and the nontoxic reverse sequence A$\beta$P40-1 and rat amylin peptides had no effect on channel activity. In separate toxicity studies, nimodipine attenuated the toxic effects of A$\beta$P1-40 on NB cells, however, was ineffective in preventing A$\beta$P25-35 toxicity. A$\beta$P25-35 may produce its toxicity by detergent action. Since A$\beta$P25-35 does not occur normally or in pathologic samples, A$\beta$P1-40 was the primary focus of this thesis.^ In recordings using synthetic vesicles, A$\beta$P1-40 formed nimodipine-insensitive, non voltage-gated, low-conductance channels for both mono- and divalent cations. The permeability sequence, estimated from reversal potentials, was P$\rm\sb{Cs}\sp+ \u3eP\sb{K}\sp+ \u3eP\sb{Na}\sp+\geq P\sb{Ca}\sp{2+}.$ Increased Ca$\sp{2+}$ reduced the conductance of monovalent ions through A$\beta$P channels. Physiological levels of vesicle-membrane cholesterol attenuated calcium conductance and reduced the P$\sb{\rm open}.$^ These data demonstrate that A$\beta$P1-40 can increase calcium entry into neuronal cells through ion channels via both direct and indirect mechanisms. Understanding the mechanism may help in designing appropriate pharmacological therapies for Alzheimer\u27s disease.