Tauroursodeoxycholic acid prevents amyloid-beta peptide-induced neuronal death via a phosphatidylinositol 3-kinase-dependent signaling pathway

Abstract

Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates cell death by interrupting classic pathways of apoptosis. Amyloid-beta (Abeta) peptide has been implicated in the pathogenesis of Alzheimer's disease, where a significant loss of neuronal cells is thought to occur by apoptosis. In this study, we explored the cell death pathway and signaling mechanisms involved in Abeta-induced toxicity and further investigated the anti-apoptotic effect(s) of TUDCA. Our data show significant induction of apoptosis in isolated cortical neurons incubated with Abeta peptide. Apoptosis was associated with translocation of pro-apoptotic Bax to the mitochondria, followed by cytochrome c release, caspase activation, and DNA and nuclear fragmentation. In addition, there was almost immediate but weak activation of the serine/threonine protein kinase Akt. Inhibition of the phosphatidylinositide 3 prime-OH kinase (PI3K) pathway with wortmannin did not markedly affect Abeta-induced cell death, suggesting that this signaling pathway is not crucial for Abeta-mediated toxicity. Notably, co-incubation with TUDCA significantly modulated each of the Abeta-induced apoptotic events. Moreover, wortmannin decreased TUDCA protection against Abeta-induced apoptosis, reduced Akt phosphorylation, and increased Bax translocation to mitochondria. Together, these findings indicate that Abeta-induced apoptosis of cortical neurons proceeds through a Bax mitochondrial pathway. Further, the PI3K signaling cascade plays a role in regulating the anti-apoptotic effects of TUDCA.

Figures

Figure 1

Aβ-induced apoptosis in rat cortical neurons is inhibited by TUDCA. Cells cultured for 3 d were incubated either with vehicle (control), 25 μM Aβ fragment 25–35, 100 μM TUDCA, or a combination of Aβ plus TUDCA for 24 h. In co-incubation experiments, cells were pretreated with TUDCA for 12 h and the bile acid was left in the culture medium with Aβ. A: Morphological and biochemical characteristics of apoptosis in rat cortical neurons incubated with Aβ peptide. Fluorescence microscopy of Hoechst staining (top) shows condensed or fragmented nuclei indicative of apoptosis. DNA laddering (middle) and caspase-3 processing (bottom) are also characteristic of apoptotic cell death. B: TUDCA prevents Aβ-induced apoptosis in rat cortical neurons. Histograms show mean ±SEM values of nuclear fragmentation (top) and caspase-3–like activity (bottom) for at least 5 independent experiments. *P < 0.01 from control; †P < 0.01 and ‡P < 0.05 from Aβ peptide.

Figure 2

TUDCA inhibits Aβ-induced cytochrome c release in rat cortical neurons. Cells were incubated with either vehicle (control), 25 μM Aβ fragment 25–35, 100 μM TUDCA, or a combination of Aβ plus TUDCA for 24 h as described in Materials and Methods. Mitochondrial (top) and cytosolic (bottom) proteins were processed for Western blot analysis. Following SDS-PAGE and transfer, the nitrocellulose membranes were incubated with a monoclonal antibody to cytochrome c (Cyt c). Histograms are mean ±SEM for at least 3 different experiments. §P < 0.05 and *P < 0.01 from control; ‡P < 0.05 from Aβ.

Figure 3

Aβ-induced translocation of Bax and modulation of Bcl-2 proteins in rat cortical neurons. Mitochondrial, cytosolic, and total proteins were processed for Western blot analysis. Following SDS-PAGE and transfer, the nitrocellulose membranes were incubated with a monoclonal antibody to Bcl-2 and polyclonal antibodies to Bax and Bcl-xL. A: TUDCA inhibits Aβ-induced translocation of Bax. Neuronal cells were incubated with vehicle (control), 25 μM Aβ fragment 25–35, 100 μM TUDCA, or a combination of Aβ plus TUDCA for 24 h as described in Materials and Methods. Representative Western blots are shown for mitochondrial (top) and cytosolic (bottom) fractions. Histograms are mean ±SEM for at least 3 different experiments. §P < 0.05 from control; ‡P < 0.05 from AβB: Aβ markedly upregulates Bax expression but only slightly induces Bcl-2 expression. Cells cultured for 3 d were incubated with either vehicle (control), or 25 μM Aβ fragment 25–35 for 5, 15, and 30 min and 1, 8, and 24 h. Representative Western blots are shown for Bax, Bcl-2, Bcl-xL, and β-actin proteins.

Figure 4

TUDCA and Aβ modulate Akt phosphorylation in rat cortical neurons. Cells were incubated with vehicle (control), 25 μM Aβ fragment 25–35, or 100 μM TUDCA for 5, 15, and 30 min and 1, 8, and 24 h as described in Materials and Methods. After incubation with Aβ (A) or TUDCA (B), total proteins were processed for Western blot analysis. Following SDS-PAGE and transfer, the nitrocellulose membranes were incubated with a polyclonal antibody for p-Akt1. Results were normalized to total Akt1/2 protein expression. Histograms are mean ±SEM for at least 3 different experiments. §P < 0.05 and *P < 0.01 from respective control.

Figure 5

TUDCA prevents Aβ peptide–induced apoptosis through a PI3K-dependent pathway. Cultured neurons were incubated with either vehicle (control), 25 μM Aβ fragment 25–35, 100 μM TUDCA, or a combination of Aβ plus TUDCA, as described in Materials and Methods. Cells were pretreated with 200 nM wortmannin for 1 h prior to adding TUDCA, and the PI3K inhibitor was left in the culture medium during incubation with both Aβ and TUDCA. A: Wortmannin slightly increased Aβ toxicity at 24 h, but almost completely abolished the inhibitory effect of TUDCA in Aβ-induced apoptosis. Histograms show mean ±SEM values of nuclear fragmentation for at least 5 different experiments. *P < 0.01 from controls treated with wortmannin. B: PI3K inhibition was associated with reduced Akt phosphorylation by Aβ at 5 min, TUDCA alone, or a combination of Aβ plus TUDCA. After incubation with wortmannin, total proteins were processed for Western blot analysis. Following SDS-PAGE and transfer, the nitrocellulose membranes were incubated with polyclonal antibodies for p-Akt1 and total Akt1/2. Representative Western blots are shown for Akt phosphorylation. C: Wortmannin almost completely abolished the inhibitory effect of TUDCA on Aβ-mediated Bax translocation from cytosol to mitochondria at 24 h. After incubation with wortmannin, total, cytosolic, and mitochondrial proteins were processed for Western blot analysis. Following SDS-PAGE and transfer, the nitrocellulose membranes were incubated with antibodies for p-Bad and Bax. Histograms are mean ±SEM for at least 3 independent experiments. §P < 0.05 and *P < 0.01 from controls treated with wortmannin.