Peroxisome-proliferator-activated receptor gamma induces a clearance mechanism for the amyloid-beta peptide

Abstract

We investigated whether peroxisome proliferator-activated receptor gamma (PPARgamma) could be involved in the modulation of the amyloid cascade causing Alzheimer's disease. Inducing expression or activating PPARgamma using synthetic agonists of the thiazolinedione family results in a dramatic decrease in the levels of the amyloid-beta (Abeta) peptide in the conditioned medium of neuronal and non-neuronal cells. PPARgamma does not affect expression or activity of any of the secretases involved in the generation of the Abeta peptide but induces a fast, cell-bound clearing mechanism responsible for the removal of the Abeta peptide from the medium. Although PPARgamma expression is generally low in the CNS, induction of PPARgamma expression during inflammation could be beneficial for inducing Abeta clearance. We confirm that the Abeta clearance mechanism can indeed be induced by PPARgamma activation in primary murine-mixed glia and cortical neuronal cultures. Our results suggest that PPARgamma-controlled mechanisms should be explored further as potential drug targets for Alzheimer's disease treatment.

Figure 1.

Analysis of the processing of APP in hPPARγ-overexpressing cells. HEK293 APPsw cells were transduced with recombinant adenovirus (MOI 100) driving expression of GFP (lanes 1, 2), hPPARγ (lanes 3, 4), hAPP695sw (lanes 5, 6), or APP-C99 together with GFP (lanes 7, 8) or hPPARγ (lanes 9, 10). Cells and conditioned medium were collected 16 hr after infection. A, Western blot detection of secreted Aβ with WO2 antibody. B, Western blot detection of APPsα with 6E10 antibody. C, Western blot detection of APPsβsw with 54 antibody. D, E, Full-length APP, β-APP-CTF, and α-APP-CTF were detected by Western blot using B63 antibody. F, Levels of endogenous β-actin are shown as a loading control for the cell extracts. G, Cells were transduced with adenovirus driving expression of either GFP or hPPARγ with different MOI (1, 3, 10, 30, and 100). Aβ levels were detected with WO2 antibody.

Figure 2.

Analysis ofγ-secretase cleavage of APP and of Notch. A, Membranes from HEK293 APPsw cells expressing GFP (lanes 1, 2) or hPPARγ (lanes 3, 4) were purified and incubated without (lanes 1, 3) or with (lanes 2, 4) APP C100-flag substrate at 37°C for 16 hr. Aβ was detected with WO2 antibody. hPPARγ does not affect Aβ generation in this assay. B-D, HEK293 APPsw cells were coinfected with mNotchΔE-myc and either GFP (lanes 2, 3) or hPPARγ adenovirus (lanes 4, 5). Cells coinfected with NICD and GFP (lane 6) are shown as a positive control for the detection of NICD. Cells only infected with GFP (lane 1) are used as a negative control. Cells were treated for 4 hr with lactacystin (10 μm) to avoid proteasome degradation of NICD. B, Detection of Notch ΔE-myc and NICD using an anti-myc antibody. Levels of the substrate NotchΔE and NICD were comparable in GFP- and hPPARγ-transduced cells, showing a normal processing by γ-secretase. C, Detection of NICD with antibody (val 1744) specific for the cleaved form of Notch. D, Conditioned medium was collected, and Aβ levels were detected with WO2 antibody to confirm the activity of PPARγ in this experiment.

Figure 3.

Aβ intracellular accumulation is decreased after overexpression of hPPARγ. HEK293 APPsw cells were transduced with GFP or hPPARγ recombinant adenovirus. At four different postinfection time points, conditioned medium was collected and cells were lysed in 1% Triton buffer. A, Detection of Aβ and APPsα was performed using WO2 antibody. B, Total cell extract was subjected to immunoprecipitation with B7/8 antibody and immunoblotted with WO2 for intracellular Aβ detection. Both secreted and intracellular Aβ were downregulated in hPPARγ cells.

Figure 4.

Levels of secreted Aβ from GFP-transduced cells are not changed after incubation with conditioned medium from hPPARγ cells. A, HEK293 APP695sw cells were infected with GFP or hPPARγ adenovirus. Sixteen hours after infection, medium was collected. Medium from GFP-transduced cells was incubated with fresh medium (lane 1) or with hPPARγ medium (lane 2) at 37°C for 2 hr. Aβ stability was detected by immunoprecipitation (B7/8 antibody) and immunoblot analysis (WO2 antibody). As additional controls, we also included 1% FBS medium (lane 4) and medium from hPPARγ-transduced cells (lane 3) with no Aβ detectable, as expected. B, HEK293 cells were cotransduced with hAPP695sw and GFP adenovirus. After 48 hr of infection, cells were incubated with medium taken 16 hr after infection from hPPARγ-transduced HEK293 cells (lanes 1-4), medium taken 16 hr after infection from GFP-transduced HEK293 cells (lanes 5-8), or with 1% FBS DMEM fresh medium (lanes 9-12) for the times indicated. Samples from media were subjected to immunoblot analysis with WO2 antibody to detect APPsα and Aβ. HEK293 nontransduced with hAPP695sw did not have detectable levels of Aβ in the medium (lanes 1, 5). There were no changes in Aβ stability after incubation of GFP cells with the medium from hPPARγ cells.

Figure 5.

Aβ₄₀ peptide is rapidly cleared from medium of cells expressing hPPARγ. HEK293 cells were infected with either GFP or hPPARγ adenovirus. Aβ₄₀ synthetic peptide was added to the medium 16 hr after infection. Conditioned medium was collected at 7 min, 20 min, 1 hr, 3 hr, or 9 hr, and Aβ was detected by Western blot with WO2 antibody. Endogenous levels of secreted Aβ are not detectable in this assay (Fig. 4 B, lanes 1, 5). The levels of the Aβ in the medium decrease gradually with time in hPPARγ-overexpressing cells. B, Logarithmic representation of two experiments. The medium value of Aβ from all GFP-transduced cells is set as 100% and considered as an initial amount. All values refer to the initial amount.

Figure 6.

Downregulation of Aβ levels is a specific effect of the hPPARγ isoform of the PPAR family. HEK293 APPsw cells were infected either with GFP, hPPARγ, hPPARα, hPPARδ, or hRXR recombinant adenoviruses. A, Samples from medium were analyzed by Western blot, and APPsα and Aβ were detected with WO2 antibody. B-D, Levels of expression of hRXR, hPPARγ, and hPPARδ were detected by Western blot from nuclear extracts using specific antibodies. E-G, Levels of expression of the SP1 transcription factor are detected as a loading control. The results show that Aβ is downregulated specifically after hPPARγ overexpression.

Figure 7.

Downregulation of Aβ is reached through TZD activation of endogenous PPARγ. A-D, HEK293 APPsw cells were transduced with GFP adenovirus and incubated for 16 hr with several TZD drugs or only transduced with hPPARγ adenovirus. Levels of APPsα and Aβ were detected by Western blot (WO2 antibody) from samples of conditioned medium. A, Cells were treated with 1, 3, 10, and 30 μm troglitazone with DMSO (solvent) or transfected with hPPARγ without treatment. B, Cells were treated with 1, 3, 10, and 30 μm pioglitazone with DMSO or transfected with hPPARγ. C, Cells were treated with 30 μm 9-cis-RA with DMSO or transfected with hPPARγ. D, Cells were treated with 30 μm rosiglitazone, ethanol (solvent), or transfected with hPPARγ. A-D, Aβ quantification from two to four independent experiments. Values of Aβ were normalized for APPsα (Aβ/APPsα) and referred to the GFP control value set as 100%.

Figure 8.

The PPARγ antagonist GW9662 effectively blocks the TZD effect on Aβ levels. HEK293 APPsw cells are incubated for 16 hr either with troglitazone (1, 3, and 10 μm) alone or together with GW9662 (1 μm). DMSO (solvent) treatment shows the control levels of Aβ. Levels of APPsα and Aβ were detected by Western blot (WO2 antibody) from samples of conditioned medium.

Figure 9.

Aβ is downregulated in differentiated human neuroblastoma IMR-32 cells in primary murine-mixed glial cultures and in primary cortical cultures after hPPARγ induction. A, Differentiated IMR-32 cells were infected with either GFP or hPPARγ recombinant adenovirus and metabolically labeled with ³⁵S-methionine for 5 hr. Aβ was immunoprecipitated from medium with 4G8 antibody and analyzed by phosphorimaging. B, Primary murine-mixed glial cultures are cotransduced with APPsw and either GFP or hPPARγ recombinant adenovirus. Secreted Aβ was detected with WO2 antibody. C, Primary cortical cultures are transduced with APPsw together with hPPARγ and treated with increasing concentrations of troglitazone (3, 10, and 30 μm).