Potassium Depolarization and Raised Calcium Induces alpha-Synuclein Aggregates

作者：Follett Jordan; Darlow Bonnie; Wong Mathew B; Goodwin Jacob; Pountney Dean L^*

来源：Neurotoxicity Research, 2013, 23(4): 378-392.

DOI：10.1007/s12640-012-9366-z

• alpha-Synuclein
• Parkinson%26apos
• s disease
• Calcium

alpha-Synuclein is the key aggregating protein in Parkinson%26apos;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 (%26gt; 2 mu m), perinuclear alpha-synuclein aggregates or multiple smaller (%26lt; 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 %26gt; 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.

• 出版日期2013-5