Astrocytes protect neurons from Aβ1-42 peptide-induced neurotoxicity increasing TFAM and PGC-1 and decreasing PPAR-γ and SIRT-1

Abstract

One of the earliest neuropathological events in Alzheimer's disease is accumulation of astrocytes at sites of Aβ1-42 depositions. Our results indicate that Aβ1-42 toxic peptide increases lipid peroxidation, apoptosis and cell death in neurons but not in astrocytes in primary culture. Aβ1-42-induced deleterious neuronal effects are not present when neurons and astrocytes are mixed cultured. Stimulation of astrocytes with toxic Aβ1-42 peptide increased p-65 and decreased IκB resulting in inflammatory process. In astrocytes Aβ1-42 decreases protein expressions of sirtuin 1 (SIRT-1) and peroxisome proliferator-activated receptor γ (PPAR-γ) and over-expresses peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) and mitochondrial transcription factor A (TFAM), protecting mitochondria against Aβ1-42-induced damage and promoting mitochondrial biogenesis. In summary our data suggest that astrocytes may have a key role in protecting neurons, increasing neural viability and mitochondrial biogenesis, acquiring better oxidative stress protection and perhaps modulating inflammatory processes against Aβ1-42 toxic peptide. This might be a sign of a complex epigenetic process in Alzheimer's disease development.

Keywords: Alzheimer's Disease; MnSOD; NF-κB.; PGC-1; PPAR-γ; TFAM.

Figure 1

Aβ_1-42 induces loss of cell viability in neurons but not in astrocytes and in mixed-culture cells. Cell viability was determined by MTT assay in cells treated for 24 h with 5 μM Aβ_40-1 (control, C) or 5 μM Aβ_1-42 (Aβ). Data are mean ± SD of four independent experiments. *P < 0.05 vs. control.

Figure 2

In vitro DEVDase caspase-3 activity assay in astrocytes, neurons and mixed-culture cells. DEVDase caspase-3 activity was evaluated after 24 h incubation with 5 μM Aβ_40-1 as a control (C) or Aβ_1-42 as a toxic peptide (Aβ) in neurons, astrocytes and in mixed-culture cells. The results are representative of four independent experiments in quadruplicate. *P < 0.04 vs. control cells. Western-blot with Cytochrome c was evaluated in primary culture neurons. Data are mean ± SD of five independent experiments. *P < 0.05 vs. control.

Figure 3

MDA levels in neurons, astrocytes and mixed-culture cells. Cells were treated for 24 h with 5 μM Aβ_40-1 (control, C) or 5 μM Aβ_1-42 (Aβ). Data are mean ± SD of three different experiments. *P < 0.05 vs. control.

Figure 4

Aβ_1-42 increases mitochondrial aggregation in astrocytes in primary culture. Mitochondrial aggregation induced by Aβ_1-42 was determined by confocal microscopy in astrocytes in primary culture treated for 6 h with 5 μM Aβ_40-1 (C) or 5 μM Aβ_1-42 (Aβ). Fluorescence products used were: Mitotracker (250 nM) to stain mitochondria and Hoechst 33342 (2 μg ml^-1) to stain nuclei (A, B). Contrast image (C, D). Immunofluorescence was used with Tom 20 (E, F; 1:1000). Bar represents 20 μm.

Figure 5

Increase of peroxide levels, p-65 subunit (NF-κB), MnSOD, and IκB expressions caused by Aβ_1-42 in astrocytes in primary culture. Peroxide levels were determined in cells treated for 24 h with 5 μM Aβ_40-1 (C) or 5 μM Aβ_1-42 (Aβ). Data are mean ± SD of three different experiments. *P < 0.01 vs. control. p-65, MnSOD and IκB protein expressions were determined in control conditions(C) and after addition of 5 μM toxic peptide Aβ_1-42 during 24 h (Aβ). Data are mean ± SD of four independent experiments. *P < 0.05 vs. control.

Figure 6

Aβ_1-42 increases TFAM and PGC-1 and decreases PPAR-γ and SIRT-1 protein expressions in astrocytes in primary culture. Astrocytes were incubated with 5 μM of Aβ_1-42 (Aβ) or 5 μM Aβ_40-1 (C) for 24 h and collected to determine TFAM, PGC-1, PPAR-γ and Sirt-1 protein expressions by western blot. A representative immunoblot is shown in the top panel. Data are mean ± SD of four independent experiments. *P < 0.05 vs. control.

Figure 7

Signalling in astrocytes in primary culture after Aβ_1-42 addition. The axis PGC-1-TFAM-Mitochondrial Biogenesis is regulated by PPAR-γ, NF-κB, SIRT-1 and MnSOD. Aβ_1-42 toxic peptide decreases SIRT-1 and PPAR-γ and increases MnSOD and NF-κB leading to PGC-1-TFAM up-regulation resulting in mitochondrial biogenesis.