Abstract/Summary

Alzheimer’s disease (AD) is the most frequently occurring form of dementia worldwide. Calcium (Ca2+) is important in maintaining cellular physiology with roles in gene expression, cell proliferation, differentiation and survival. One mechanism by which aggregated amyloid-b (Ab), a main protagonist in AD, can cause Ca2+ dysregulation and subsequent changes in cell activity is through changes in both the expression and activity of VGCCs. Carbon monoxide (CO) acting as a gasotransmitter has shown a range of cytoprotective roles potentially mediated by ion channel-dependant mechanisms. The aim of this study was to identify the effect of protofibrillar Ab1-42 on voltage-gated calcium channels (VGCCs) and to investigate how CO can modulate these channels and therefore interfere with Ab-mediated cytotoxicity. Chemical analysis and neuroblastoma activity assays revealed time-dependent (48hr) protofibril formation of Ab1-42 using NH4OH demonstrating protofibril formation that structurally and functionally resembles aggregated Ab1-42 cytotoxicity seen in vivo. Moreover, mimicking the microenvironment of cell membranes, SDS was shown to aid Ab1- 42 aggregation. Molecular approaches (qPCR) revealed that within retinoic acid (RTA) differentiated cells protofibril Ab1-42 changes Cav1 expression at gene level, proposed to be representative of Ca2+ dysregulation seen at early stages of AD. On the other hand, electrophysiology findings revealed that Ab1-42 is capable of inhibiting Cav2.2 currents of stably transfected HEK293 cells. Reduction in Cav2.2 dependent Ca2+ influx represents changes in Ca2+ homeostasis seen at late stages of AD where significant neurotransmitter depletion and synaptotoxicity is observed. CO, generated using the donor CORM2 (tricarbonyldichlororuthenium(II) dimer), was shown to inhibit Cav2.2 channels through selective changes in channel kinetics. Further through electrophysiological investigation it was revealed that CO acts similarly to an oxidant, with the reducing agent DTT (dithiothreitol) reversing CO inhibitory effects on the channel. Findings from the current study aid our understanding of AD by highlighting the different effects of Ab on Ca2+ homeostasis dependent on early or late AD stage. Moreover, this study reveals that CO, might inhibit VGCCs, via an oxidant action, which may be beneficial in targeting Ca2+ dyshomeostasis seen at early stage of the disease where neuronal hyperactivity is observed. This study demonstrates the potential of CO, or the activation of CO-dependent pathways as a novel therapeutic against AD.