Potassium Depolarization and Raised Calcium Induces α-synuclein Aggregates

Neurotox Res. 2013 May;23(4):378-92. doi: 10.1007/s12640-012-9366-z. Epub 2012 Dec 19.

Abstract

α-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 α-synuclein oligomers. Previous post-mortem studies have shown relative sparing of neurons in PD that are positive for the Ca(2+) buffering protein, calbindin, and recent cell culture and in vitro studies have shown that α-synuclein aggregation can be induced by Ca(2+). We hypothesized that depolarization with potassium resulting in raised Ca(2+) in a PD cell culture model will lead to the formation of α-synuclein protein aggregates and that the intracellular Ca(2+) 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/α-synuclein cells. Raised intracellular free Ca(2+) 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 μm), perinuclear α-synuclein aggregates or multiple smaller (<2 μm), cytoplasmic accumulations. Cells pre-treated with varying concentrations of trimethadione (TMO), a calcium channel blocker, showed suppression of the Ca(2+) transient following KCl treatment and no α-synuclein aggregates at TMO concentrations >5 μM. Quantitative analysis revealed a significant increase in the number of cells bearing α-synuclein cytoplasmic inclusions in both HEK293T/α-synuclein and SHSY-5Y cells when transient intracellular raised Ca(2+) was induced (p = 0.001). BAPTA-AM pre-loading significantly suppressed α-synuclein aggregates (p = 0.001) and the intracellular free Ca(2+) transient. This study indicates that raised intracellular Ca(2+) mediated by K(+) depolarization can lead to α-synuclein aggregation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / metabolism*
  • Cell Line, Tumor
  • HEK293 Cells
  • Humans
  • Intracellular Fluid / chemistry
  • Intracellular Fluid / drug effects
  • Intracellular Fluid / metabolism
  • Potassium Chloride / metabolism*
  • Potassium Chloride / pharmacology
  • alpha-Synuclein / metabolism*

Substances

  • alpha-Synuclein
  • Potassium Chloride
  • Calcium