Figure 7.
Modulation of ATP13A2 expression reduces basal intracellular calcium concentration. (A) Rat primary cortical neurons were co-transfected with V5-tagged human ATP13A2 (or control empty vector) and DsRed plasmids at a 10:1 molar ratio at DIV 9 to label transfected neurons. At DIV 12, neurons were loaded with Fluo-4 AM fluorescent dye to monitor the intracellular calcium levels of individual DsRed-positive neurons by time-lapse, live-cell confocal microscopy. Representative time-lapse series of baseline intracellular calcium levels (over 120 s) and following acute cadmium exposure (over 240 s) in DsRed-positive neurons. Warmer tones indicate higher intracellular calcium concentrations as indicated by lower color scale bar. (B) ATP13A2 overexpression reduces the levels of basal and cadmium-induced intracellular calcium in neurons. (C) Time course of average intracellular calcium levels induced by cadmium exposure (at time 0 s) expressed as increased calcium levels over initial basal levels for each neuron. Data were fitted to a first-order kinetic model, and the mean (D) rate (half-life, t1/2) and (E) maximum peak calcium levels were determined. (F–J) Similar experiments were conducted on primary cortical neurons co-transfected with non-silencing control shRNA or ATP13A2-specific shRNA and DsRed plasmids at a 10:1 molar ratio. (F) Representative time-lapse series of baseline and cadmium-induced intracellular calcium levels. (G) ATP13A2 silencing reduces the levels of basal and cadmium-induced intracellular calcium in neurons. (H) Time course of intracellular calcium levels induced by cadmium exposure and determination of mean (I) rate and (J) maximum calcium levels. Data represent n = 15 neurons/condition sampled from five independent experiments. **P < 0.01 or ***P < 0.005 compared with the appropriate control condition as indicated by lines, or by comparing basal versus cadmium conditions, assessed by unpaired Student's t-test.