Reduction of beta-amyloid levels by novel protein kinase C(epsilon) activators

Abstract

Isoform-specific protein kinase C (PKC) activators may be useful as therapeutic agents for the treatment of Alzheimer disease. Three new epsilon-specific PKC activators, made by cyclopropanation of polyunsaturated fatty acids, have been developed. These activators, AA-CP4, EPA-CP5, and DHA-CP6, activate PKCepsilon in a dose-dependent manner. Unlike PKC activators that bind to the 1,2-diacylglycerol-binding site, such as bryostatin and phorbol esters, which produce prolonged down-regulation, the new activators produced sustained activation of PKC. When applied to cells expressing human APPSwe/PS1delta, which produce large quantities of beta-amyloid peptide (Abeta), DCP-LA and DHA-CP6 reduced the intracellular and secreted levels of Abeta by 60-70%. In contrast to the marked activation of alpha-secretase produced by PKC activators in fibroblasts, the PKC activators produced only a moderate and transient activation of alpha-secretase in neuronal cells. However, they activated endothelin-converting enzyme to 180% of control levels, suggesting that the Abeta-lowering ability of these PKCepsilon activators is caused by increasing the rate of Abeta degradation by endothelin-converting enzyme and not by activating nonamyloidogenic amyloid precursor protein metabolism.

FIGURE 1.

Structures of DCP-LA, DHA-CP6, EPA-CP5, and AA-CP4.

FIGURE 2.

Activation of purified PKCϵ by PKC activators. Purified PKCα, βII, γ, δ, or ϵ (9 ng) was preincubated for 5 min at room temperature with DHA-CP6 (A), EPA-CP5 (B), AA-CP4 (C), or DCP-LA (D) followed by PKC activity measurement as described under “Experimental Procedures.” E, activation of purified PKCϵ by other cyclopropanated and epoxidized fatty acids, alcohols, and methyl esters. Samples were preincubated for 5 min at room temperature with purified PKCϵ (500 ng) followed by PKC activity measurement. Values are x̄ ± S.E., two-tailed Student's t test. *, p < 0.02; **, p < 0.001.

FIGURE 3.

Time course of PKC activation in cultured human SH-SY5Y cells (A) and cultured primary rat hippocampal cells (B). DHA-CP6, EPA-CP5, or AA-CP4 in 10 μl of ethanol was added to cells growing in 6-well plates with 1 ml of culture medium. At the indicated time, the cells were washed with phosphate-buffered saline and scraped into 200 μl of homogenization buffer followed by PKC activity measurement as described under “Experimental Procedures.” *, p < 0.02; **, p < 0.001.

FIGURE 4.

Decreased intracellular levels of Aβ in cells (A) and in culture medium (B) from cells exposed to PKC activators. Neuro2a neuroblastoma cells transfected with human APPSwe/PS1Δ9 (23, 24), growing in 6-well plates, were incubated with low serum (1%) medium for 24 h. The medium was replaced with fresh low serum medium and various concentrations of bryostatin, DCP-LA, or DHA-CP6 in 10 μl of ethanol, or 10 μl of ethanol alone was added. After 18 h, the cells were washed with phosphate-buffered saline and scraped into 200 μl of homogenization buffer, homogenized, and Aβ was measured by ELISA. The medium was collected, and Aβ was isolated by adsorption onto reversed phase columns (Waters C₁₈ Sep-Pak cartridges), washed, and eluted with acetonitrile followed by ELISA measurements.

FIGURE 5.

Effect of DHA-CP6 on degradation of exogenously applied Aβ in human SH-SY5Y neuroblastoma cells. Various concentrations of DHA-CP6 in 10 μl of ethanol or 10 μl of ethanol alone were added to cells grown in 6-well plates. After 5 min, fresh Aβ1–42 (100 nm) or buffer was applied, and the cells were incubated for 24 h. Aβ1–42 was then measured in the cells and culture medium by ELISA. *, p < 0.02.

FIGURE 6.

Effect of PKC activators on TACE activity in SH-SY5Y neuroblastoma cells (A) and cultured primary rat hippocampal neurons (B). DHA-CP6, EPA-CP5, or AA-CP4 in 10 μl of ethanol or 10 μl of ethanol alone was added at a final concentration of 1 μm to cells grown in 12- or 24-well plates. After various periods of time, the cells were collected, and TACE activity was measured fluorometrically. *, p < 0.02.

FIGURE 7.

Activation of ECE by PKC activators in SH-SY5Y cells (A) or cultured neurons (B). Bryostatin (Bryo; 0.27 nm), DCP-LA (1 μm), DHA-CP6 (1 μm), EPA-CP5 (1 μm), AA-CP4 (1 μm), or ethanol alone was added to cells growing on 12- or 24-well plates. After various periods of time, the cells were collected, and ECE activity was measured fluorometrically. *, p < 0.05; **, p < 0.001.

FIGURE 8.

Lack of activation of purified ECE by PKC activators. ECE activity was measured as described under “Experimental Procedures” in the presence of recombinant ECE (1784ZN, 20 ng; R&D Systems) and varying concentrations of DHA-CP6, EPA-CP5, or AA-CP4 for 60 min at 37 °C.

FIGURE 9.

Lack of effect of DCP-LA and DHA-CP6 on cell survival and cell proliferation. Various concentrations of DCP-LA or DHA-CP6, or ethanol alone were added to SH-SY5Y cells growing on 6-well plates. Cell viability was measured by adding alamarBlue reagent (Invitrogen) to 10% of the culture volume. AlamarBlue is chemically reduced in viable cells to form a fluorescent product. The fluorescence was measured in a microplate reader at 1, 2, 3, and 4 h, and the slope of the curve of fluorescence intensity versus time was calculated. Cell proliferation was measured by adding 100 μl of CyQuant NF, which is a cell-permeable DNA-binding dye. After 60 min, the fluorescence, which is proportional to the number of cells, was measured in a microplate reader.