Figure 1

From: Amyloid proteotoxicity initiates an inflammatory response blocked by cannabinoids

Figure 1

Intraneuronal Aβ induces an inflammatory response that is potentiated by arachidonic and linoleic acids. (a) MC65 cells were induced to make intracellular Aβ by the removal of tet (tet−) from the culture medium in the presence (SI+) or absence (SI−) of 10 μmol/l γ-secretase inhibitor 10 (Calbiochem) and cell death assayed on day 4. (b). Expression of intracellular Aβ using 6E10 antibody on day 2 in same conditions as in a. Arrow indicates C99 and the 100 kDa band is APP. (c) MC65 cells were induced to make Aβ (−tet) or uninduced (+tet) in the presence or absence of 10 μmol/l γ secretase inhibitor (SI) 1 and 2 days later proteins were assayed by western blotting. (d) Protein amount was quantified and normalized either to actin or in the case of phosphorylation to the total protein. D1=Day 1, D2=Day 2, D2S=Day 2+SI. (e) Western blot of Aβ two days after tet withdrawal (ΔT) in the presence of MK806 (MK, 1 μmol/l), ketorolac (KETO, 10 μmol/l), caspase 1 inhibitor (CPS 1, 50 μmol/l), FkG 11 (FK, 5 μmol/l), THL (5 μmol/l) or CNB-001 (CNB, 1 μmol/l). (f) Cells were incubated for 4 days in the presence of the caspase 1 inhibitor (CPS1) Z-YVAD-FMC (50 μmol/l), caspase 3 inhibitor (CPS3, 50 μmol/l), Z-DEVD-FMk (50 μmol/l) or the compounds in e. The percent viable cells is presented. *P<0.01, n=3–5. (g) MC65 cells were induced to make Aβ in the presence of increasing amounts of LA or AA and cell death was measured on day 2 instead of day 4 when the cells normally die. The control is cells without tet. All changes with AA or LA: P<0.01, n=4. (h) MC65 cells were grown in the absence of tet (ΔT) with 100 nmol/l J147 or 10 μmol/l γ secretase inhibitor (SI) and eicosanoid accumulation assayed in the culture medium 2 days later, and presented as fold change relative to uninduced cells. Baseline values (fmole/2×106 cells) are: PGE2, 49; PGF2, 1315; PGD2, 257; 5-HETE, 11; 12 HETE, 101; 15 HETE, 28. These data are from a single experiment, but similar results were obtained in three independent experiments. The absolute amounts of extracellular eicosanoids varied, but the relative changes within the individual experiments were similar (see the separate experiment in Figure 2). APP, amyloid precursor protein; Aβ, beta amyloid.