Figure 6.
Clusterin inhibits physiological damage caused by Aβ oligomers. (A) (i) The cytosolic Ca2+ concentration is shown as a function of time in astrocytes to which 500 nM (total peptide) Aβ42 (containing 19.5 nM oligomers), incubated with 500 nM clusterin, has been added; 327 astrocytes and 187 neurons were examined. (ii) The same experiment with 500 nM Aβ40 (1.5 nM oligomers). Each line represents the intracellular calcium within a single cell; 69 cells were examined, and 158 astrocytes and 84 neurons were examined. (B) (i) The percent change in rate of ROS production in astrocytes and neurons in mixed cultures upon the addition of either 500 nM Aβ42 containing 19.5 nM oligomers (termed “oligo”) or 500 nM Aβ40 containing 1.5 nM oligomers (“oligo”) or 500 nM monomeric solutions (“mono”) in the presence or absence of a 1:1 molar ratio of clusterin to Aβ (“clusterin”). ** represents a p-value <0.01, and **** represents a p-value <0.0001 using a Kruskal–Wallis test followed by Dunn’s post-test. Comparisons were performed with the “oligo” sample of the same Aβ isoform. (ii) The HEt ratio as a function of time upon the addition of Aβ42 (50 nM total peptide), Aβ42 (50 nM total peptide) mixed with 50 nM clusterin, or clusterin only (50 nM total peptide) to astrocytes and neurons in mixed primary cultures. Experiments examined 61 cells with Aβ42 and 59 with Aβ40. (iii) The HEt ratio as a function of time upon the addition of Aβ42 (500 nM total peptide containing 19.5 nM oligomers) mixed with 0, 0.5, 50, or 500 nM of clusterin and then added to astrocytes and neurons in mixed primary cultures. For the various ratios of (Aβ/clusterin), the following number of cells was examined: 10:1, 111; 100:1, 99; and 1000:1, 114. (C) (i) Fluorescence of the NucView488 caspase-3 substrate as a function of time following the treatment of primary cultures containing both neurons and astrocytes with a mixture of 500 nM Aβ42, containing 19.5 nM oligomers, and 500 nM clusterin; 215 cells were monitored over 4 different samples. (ii) The proportion of cells (%) that experience activation of caspase-3 following 30 min of incubation with either oligomeric Aβ42 solutions (total peptide concentrations of 50 nM and 500 nM with oligomer concentrations of 1.95 nM and 19.5 nM) or Aβ42 solutions (500 nM total peptide, 19.5 nM oligomers) incubated with 500 nM clusterin. These data are the end points (after 30 min) of the experiments presented in Figure 4A and panel C, i. ** signifies a p-value <0.01 by a Mann–Whitney nonparametric rank correlation. Comparisons were performed relative to data acquired with 500 nM oligomers of Aβ42. (D) (i) The fEPSP slope as a function of time in hippocampal slices incubated prior to each experiment for 2 h with a mixture of 500 nM Aβ42 (containing 19.5 nM oligomers) and 500 nM clusterin. The induction of LTP is observed after HFS. Data were recorded for 9 slices. (ii) The fEPSP slope as a function of time following tetanus in hippocampal slices incubated prior to the experiment for 2 h with a mixture of 500 nM Aβ42 (containing 19.5 nM oligomers). This experiment was conducted as a biological control for the results presented in panel D, i. No induction of LTP is observed, and 4 slices were studied.