Korol T. Influence of beta-amyloid peptide on calcium signalling in hippocampal cell culture of rats

This dissertation is devoted to the investigation of influence of the beta-amyloid peptide, main component of senile plaques in the brain of patients with Alzheimer's disease, on calcium signalling in hippocampal cell culture. Accumulation of beta-amyloid peptide of 42 amino acid length (Abeta(1-42)) in brain is an important characteristic for the etiology of Alzheimer's disease. Of all the possible processes generating the neurotoxic effects by Abeta(1-42), disruption of intracellular Ca2+ homeostasis is the primary event. The aim of the study was to discover changes in basal calcium level and mitochondrial function as well as alterations in properties of voltage-gated calcium channels in hippocampal cell culture with experimental induced Alzheimer's disease. The changes in neuronal Ca2+ homeostasis and voltage-dependent characteristics of calcium channels were revealed in rat hippocampal cell culture at comparison of Ca2+ signalling in control conditions and after 24-hour treatment with Abeta(1-42). The cytoplasmic free Ca2+ concentration ([Ca2+]i) was measured using fura-2 based microfluorometry. Hyperpotassium solution was used for inducing Ca2+ transients. To compare mitochondrial contribution to Ca2+ signals in experimental conditions was used uncoupler of mitochondria CCCP (10 mkM) dissolved in hyperpotassium solution. It was revealed that after 24-hour incubation of hippocampal cell culture with Abeta(1-42) basal level of [Ca2+]i was 138±12 nM vs. 76±8 nM in control conditions (P < 0.01, n = 19). Amplitude of Ca2+ transient in control conditions was 506±20 nM, and after treatment with Abeta(1-42) this parameter increased to 622±34 nM (P < 0.05, n = 18). Amplitude of transients induced by hyperpotassium solution with addition of CCCP was 23±3% greater than amplitude of transient induced by hyperpotassium solution alone in control cells. Area of transient that corresponds to amount of cell calcium after incubation of hippocampal cell culture with beta-amyloid peptide was greater than control transient area. After incubation with Abeta(1-42) difference between amplitudes induced by hyperpotassium solution with and without CCCP was not detected that can point to impairment of Ca2+ uptake function of mitochondria during maximal increasing of [Ca2+]i. Moreover, in cells treated with Abeta(1-42) Ca2+ transients induced by hyperpotassium solution with CCCP were higher and broader than that ones in control neurons. Heightened basal level of [Ca2+]i after incubation of cells with Abeta(1-42) may cause to intensification of Ca2+ uptake by mitochondria in cell resting state that may result in overload of organelles. It seems that this may be the reason for impossibility of rapid absorption of Ca2+ by mitochondria during excitation of the cell. Overload of mitochondria with Ca2+ may result in triggering of apoptosis. Current-voltage relationship, steady-state activation and steady-state inactivation curves of voltage-operated calcium channels of hippocampal cell culture were obtained using whole-cell modification of patch clamp technique. Current density in A?-treated cells was 70 % higher (P < 0.05, n = 12) than in control ones. Current-voltage relation of calcium channels after incubation of hippocampal cell culture with Abeta(1-42) had 10-mV shift to the hyperpolarization direction in comparison with control neurons. Steady-state activation curve of calcium channels in hippocampal cells modified by Abeta(1-42) had 12-mV shift (P < 0.001, n = 10) to more negative values of membrane potential as compared to control curve. In contrast with first two voltage dependencies steady-state inactivation curve of voltage-gated calcium channels was not changed after action of Abeta(1-42). Shifts of current-voltage characteristic and steady-state activation curve of voltage-operated calcium channels to the hyperpolarization direction after cell treatment with Abeta(1-42) may point to facilitation in activation of calcium channels. Enhancement of current density after incubation of hippocampal cell culture with Abeta(1-42) lets us suggest that probability of channel open state increases. It is possible that hyperphosphorilation of calcium channel proteins by set of protein kinases induced by action of Abeta(1-42) may be the reason of such changes in voltage dependencies of calcium channels. We conclude that Abeta(1-42) affects voltage-gated calcium channels of hippocampal cell culture by means of some intermediate mechanisms or directly that leads to disturbance in cell calcium homeostasis. Such disturbances in functioning of voltage-gated calcium channels as well as mitochondria may be essential component of pathological alterations of hippocampal functions at Alzheimer's disease. Key words: hippocampal cell culture, beta-amyloid peptide, Alzheimer's disease, calcium channels, mitochondrion.