Regular articleCeramide-induced neuronal apoptosis is associated with dephosphorylation of Akt, BAD, FKHR, GSK-3β, and induction of the mitochondrial-dependent intrinsic caspase pathway
Introduction
Apoptotic neuronal cell death has been implicated in the pathology of head injury, spinal cord injury, and cerebral ischemia, as well as in chronic neurodegenerative disorders such as Alzheimer’s disease and Huntington’s disease Bredesen 2000, Eldadah and Faden 2000. Caspases are a class of proteases which play a critical role in many models of neuronal apoptosis, both in vitro and in vivo Benchoua et al 2001, Eldadah and Faden 2000, Oppenheim et al 2001, Troy et al 2001, Yakovlev and Faden 2001. There are at least two major caspase pathways: an intrinsic pathway initiated by the release of cytochrome c from mitochondria resulting in the activation of caspase-9, and an extrinsic pathway initiated by ligation of receptors of the TNFR superfamily, resulting in the activation of caspase-8 Chinnaiyan and Dixit 1997, Li et al 1997, Zou et al 1997, Zou et al 1999. One of the mechanisms proposed for initiation of apoptosis through the intrinsic pathway is activation of proapoptotic Bcl-2 family members, such as BAD, followed by mitochondrial permeabilization and cytochrome c release Luo et al 1998, Zha et al 1996.
Ceramide is a product of sphingolipid metabolism which induces a variety of cellular changes, including cell death and differentiation. A number of known apoptotic initiators, such as growth factor withdrawal, cytokines Cifone et al 1994, Hannun 1996, and cytotoxic agents (e.g., chemotherapeutic agents), activate sphingolipid metabolism causing increased ceramide levels correlated to subsequent cell death (Hannun, 1996). Exogenous application of ceramide analogs causes apoptosis in primary cerebellar granule cell (CGC) cultures as indicated by DNA fragmentation assays (Saito et al., 1998), as well as increased caspase-3 activity (Marks et al., 1998). Inhibition of either transcription or translation prevents these effects, suggesting that both RNA and protein synthesis are required for ceramide-induced apoptosis (Taniwaki et al., 1999). Application of ceramide increases intracellular caspase-3 levels and causes apoptosis of both cortical neurons and astrocytes (Keane et al., 1997). We have also found that increased ceramide levels are associated with induction of apoptosis by trophic withdrawal or etoposide, both in CGCs and in cortical neurons (Toman et al., 2002).
Ceramide may act through a variety of signaling molecules to modulate cell death Goswami and Dawson 2000, Hannun and Luberto 2000, inhibiting the PI3K antiapoptotic/prosurvival pathway by modulating the activity of Akt. The ability of Akt to promote cell survival is based on its ability to phosphorylate on residues necessary for their inactivation several proapoptotic proteins, including BAD Cardone et al 1998, del Peso et al 1997, Forkhead family transcription factor (FKHR) (Brunet et al., 1999), and—in some species—caspase-9, leading to inhibition of caspase-3 activation Cardone et al 1998, Datta et al 1997.
Mitochondria represent another potential target for ceramide-induced signaling pathways (Kroesen et al., 2001). Release of cytochrome c from the intermembrane space of mitochondria is critical for the activation of the intrinsic caspase pathway (Li et al., 1997). Such a release may reflect opening of the mitochondrial permeability transition pore, decreasing mitochondrial membrane potential and subsequent swelling/disruption of mitochondrial membranes. In a model of ceramide-dependent apoptosis, both extensive mitochondrial swelling and a decrease in the mitochondrial membrane potential have been demonstrated (Kroesen et al., 2001).
Here, we examine the mechanism of ceramide-induced apoptosis in primary cultures of rat cortical neurons. We show that changes in Akt activity and mitochondrial membrane potential were induced early after ceramide treatment, leading to activation of the intrinsic caspase pathway, and that inhibition of such mitochondrial changes reduces ceramide-induced apoptosis.
Section snippets
Results
The short acyl-chain C2 ceramide induces neuronal cell death and caspase-3-like activation. The effects of C2 ceramide on cell death and caspase activity are specific, as we could detect no changes when the cells were treated with the inactive ceramide analog (Figs. 1A and 1B, respectively). Long-chain ceramides such as C16 ceramide are also able to induce cell death and caspase-3-like activity to a degree similar to that of C2 ceramide (Figs. 1C and 1D).
During a time course of C2 treatment
Discussion
Ceramide consists of a long-chain sphingoid base with an amide-linked fatty acid component and can be synthesized via a de novo pathway by a multistep process that takes place in the endoplasmic reticulum (Futerman, 1998). Ceramide can also be generated from the degradation of sphingomyelin, complex sphingolipids and sphingosine by sphingomyelinases (SMases), cerebrosidases, and ceramidases, respectively. Due to its extremely hydrophobic nature, naturally occurring ceramide is difficult to use
Materials
The following chemicals were used: Bongkrekic acid (Sigma-Aldrich, St. Louis, MO, USA, No. B6179), PD-98059 (Biomol Research Laboratories, Plymouth Meeting, PA, USA, No. EI-360), C2-ceramide (Biomol, No. SL-100), C2-dihydroceramide (Biomol, No. SL-101), and LY-294002 (Biomol, No. ST-420). The following antibodies, obtained from Cell Signaling Technology (Beverly, MA, USA), were used: Cleaved caspase-3 No. 9661, Phospho-Akt (Ser473) No. 9271, Phospho-Akt (Thr308) No. 9275, Phospho-BAD (Ser112)
Acknowledgements
This work was supported by NIH RO1 NS36537, DAMD17-99-2-9007, and NIH HD40677. The authors thank Dr. Fen-Yang Sun at the Shanghai Medical Center for input during the early development of the project and Elvira Dabaghyan for expert technical support.
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