Figure 8

Expression of pro-aggregant Tau ^RDΔ impairs Ca ⁺⁺ regulation in primary hippocampal cell culture and N2a cells. (a) Intracellular Ca⁺⁺ concentrations in primary hippocampal cell culture transfected with GFP only (white arrow) before membrane depolarization by high KCl. Ca⁺⁺ levels are depicted by false color coding according to Fura2 intensity (scale bar in (d)). (b) Same neuron as in (a) after high KCl stimulation. (c) Intracellular Ca⁺⁺ levels in a hippocampal neuron expressing pro-aggregant Tau^RDΔ and GFP (white arrow). (d) Same neuron as in (c) after membrane depolarization. (e) Quantification of Ca⁺⁺ imaging experiments demonstrating a reduction of calcium influx after high KCl application in pro-aggregant Tau^RDΔ expressing cells. Pictures in (a)-(d) are background subtracted at the indicated location (BG1). (f) Bright field image of a N2a control cell culture not expressing pro-aggregant Tau^RDΔ. (g) Bright field image of a N2a cell culture expressing pro-aggregant Tau^RDΔ. (h) Post-hoc staining after Ca⁺⁺ imaging experiment of the same cells as in (g) by washing in 0.0001% ThioflavineS (ThS) for 5 minutes. ThS enables to distinguish between N2a cells expressing pro-aggregant Tau^RDΔ without neurofibrillary tangles (NFT, red circle), N2a cells with NFTs (green circle) and cells with NFT and destroyed membranes (white arrow). (i) Quantification of the maximum Ca⁺⁺ influx after KCl stimulation in N2a cells loaded with Fura2AM. Cell populations introduced in (f) and (g) demonstrating that cells expressing pro-aggregant Tau^RDΔ with no ThS positive staining (pro; red column) are already severely impaired but cells with visible tangle load (pro + ThS, green column) are more impaired in Ca⁺⁺ influx. N2a cells expressing anti-aggregant Tau^RDΔPP (anti, blue column) do not show reduced calcium influx compared to control cells. Values represent at least three independent experiments. Error bars represent SEM. * p-value < 0.05; ** p-value < 0.01; *** p- value < 0.001.