Inhibition of early growth factor-mediated metabolic and molecular changes by C2-ceramide: implications for commitment to apoptosis G. Arboleda, T.-J. Huang, P. Fernyhough, C. Waters, R.M. Gibson. University of Manchester.	Print Abstract Search PubMed for: Arboleda G Huang TJ Fernyhough P Waters C Gibson RM

Apoptosis has been implicated as the mechanism underlying cell death in neurodegenerative diseases including Parkinson's disease (PD) (Honig et al., 2000). Diverse endogenous and exogenous stimuli associated with neuronal death increase ceramide levels, which in turn induce apoptosis through activation and inhibition of different pathways (Hannun et al., 2000; Zhou et al., 1998). The aims of the present study were to analyse the early metabolic and molecular changes associated with C2-ceramide-induced apoptosis in a central mesencephalic cell line, CAD (Qi et al., 1997).

The metabolic response was evaluated by microphysiometry, which measures real-time changes in extracellular acidification rate (ECAR), related to variations in intracellular pH as a consequence of changes in metabolism (Hafner, 2000). Real time fluorescence microscopy was employed to monitor mitochondrial inner membrane potential by measuring resting rhodamine-123 fluorescence, and intracellular NAD(P)H levels by detecting its autofluorescence. Changes in phosphorylation status of proteins were analysed by Western blotting, and ATP levels by luminometry. Statistical analysis was performed by one-way ANOVA, followed by Tukey post-hoc test where appropriate. Results are expressed as mean ± SEM from at least three repetitions. Level of significance was set at p<0.05.

The acute response in ECAR induced by growth factors (neurotrophin-3 or IGF-1) was inhibited by C2-ceramide in a dose-dependent manner (p<0.01), and was specific, as the inactive C2-ceramide analogue, dihydroceramide, was ineffective. The ECAR response induced by growth factors was associated with increased glycolysis, determined by increased NAD(P)H autofluorescence (+32%, p<0.01) and an increase in ECAR response in response to mitochondrial uncoupling (p<0.001), and it was prevented by the Na+/H+ antiporter inhibitor, amiloride (30 µM). The metabolic changes in response to C2-ceramide coincided temporally with dephosphorylation of AKT; inhibition of the upstream kinase, PI3K, with LY294002 (10 µM), also inhibited AKT phosphorylation and the metabolic response to growth factors. Decreases in mitochondrial membrane potential (p<0.005) and ATP (p<0.02), and induction of apoptosis occurred after AKT dephosphorylation.

These results suggest that the early metabolic inhibition induced by C2-ceramide involves the PI3K/AKT pathway, and may compromise glycolysis. This may represent a new early event in the C2-ceramide-induced cell death pathway that could coordinate subsequent changes in mitochondria and commitment of cells to apoptosis.

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