Increasing Membrane Cholesterol Level Increases the Amyloidogenic Peptide by Enhancing the Expression of Phospholipase C
Figure 1
Augmentation of membrane cholesterol levels downregulated PIP2 levels and increased Aβ42. (a, b) Incubating APP-transfected HeLa cells with water-soluble cholesterol increased cholesterol levels in the plasma membrane. Cells were incubated with 0, 15, 75, and 150 μM water-soluble cholesterol for 8 h at 37°C. Filipin staining was performed for 1 h at room temperature after cholesterol enrichment. (a) A typical fluorescence image with 75 μM water-soluble cholesterol is shown in (b). Incubating the cells with water-soluble cholesterol increased the cholesterol contents in a concentration-dependent manner. Fluorescent intensities from plasma membranes were quantified as described in Section 2 (). (c) Incubating cells with water-soluble cholesterol downregulated PIP2 levels. APP-transfected HeLa cells were incubated for 8 h with 0, 15, 75, and 150 μM water-soluble cholesterol. PIP2 levels in the membrane fractions were measured by using a PIP2 ELISA kit as described in Section 2 (). (d) Incubating cells with water-soluble cholesterol downregulated PIP2 levels in time-dependent manner. Cells were incubated with 75 μM water-soluble cholesterol for 0.5, 1.5, and 5 h (). (e) Incubating cells with water-soluble cholesterol selectively increased secreted Aβ42 levels (closed bars; ). In contrast, the levels of Aβ40 were not changed by cholesterol enrichment (open bars; ). APP-transfected HeLa cells were incubated with 0, 15, 75, and 150 μM water-soluble cholesterol for 8 h. Aβ40 and Aβ42 levels were measured from the conditioned media by using ELISA method as described in Section 2. (f) Incubating cells with water-soluble cholesterol increased secreted Aβ42 levels (closed bars; ) but not Aβ40 levels (open bars; ) from neuroblastoma SH-SY5Y cells. The endogenous Aβ40 and Aβ42 levels were measured from the conditioned media by using ELISA method. *; **; ***.
