Potassium Depolarization and Raised Calcium Induces α-Synuclein Aggregates

alpha-Synuclein is the key aggregating protein in Parkinson's disease (PD), which is characterized by cytoplasmic protein inclusion bodies, termed Lewy bodies, thought to increase longevity of the host neuron by sequestering toxic soluble alpha-synuclein oligomers. Previous post-mortem studies have shown relative sparing of neurons in PD that are positive for the Ca2+ buffering protein, calbindin, and recent cell culture and in vitro studies have shown that alpha-synuclein aggregation can be induced by Ca2+. We hypothesized that depolarization with potassium resulting in raised Ca2+ in a PD cell culture model will lead to the formation of alpha-synuclein protein aggregates and that the intracellular Ca2+ buffer, BAPTA-AM, may suppress their formation. Live cell fluorescence microscopy was performed to monitor changes in intracellular free calcium in HEK293T, SH-SY5Y neuroblastoma or stably transfected HEK293T/alpha-synuclein cells. Raised intracellular free Ca2+ was consistently observed in cells treated with KCl, but not controls. Immunohistochemistry analysis on cells 48-72 h after K+ treatment revealed two subsets of cells with either large (> 2 mu m), perinuclear alpha-synuclein aggregates or multiple smaller (< 2 mu m), cytoplasmic accumulations. Cells pre-treated with varying concentrations of trimethadione (TMO), a calcium channel blocker, showed suppression of the Ca2+ transient following KCl treatment and no alpha-synuclein aggregates at TMO concentrations > 5 mu M. Quantitative analysis revealed a significant increase in the number of cells bearing alpha-synuclein cytoplasmic inclusions in both HEK293T/alpha-synuclein and SHSY-5Y cells when transient intracellular raised Ca2+ was induced (p = 0.001). BAPTA-AM pre-loading significantly suppressed alpha-synuclein aggregates (p = 0.001) and the intracellular free Ca2+ transient. This study indicates that raised intracellular Ca2+ mediated by K+ depolarization can lead to alpha-synuclein aggregation.