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The Journal of Neuroscience, September 1, 1999, 19(17):7394-7404
Mitochondrial Depolarization Is Not Required for Neuronal
Apoptosis
Aaron J.
Krohn1,
Tanja
Wahlbrink1, and
Jochen H. M.
Prehn1, 2
1 Interdisciplinary Center for Clinical Research (IZKF),
Junior Research Group "Apoptosis and Cell Death," Westphalian
Wilhelms-University, D-48149 Münster, Germany, and
2 Department of Pharmacology and Toxicology,
Philipps-University, D-35032 Marburg, Germany
Mitochondria are sites of cellular energy production but may also
influence life and death decisions by initiating or inhibiting cell
death. Mitochondrial depolarization and the subsequent release of
pro-apoptotic factors have been suggested to be required for the
activation of a cell death program in some forms of neuronal apoptosis.
We induced apoptosis in cultured rat hippocampal neurons by exposure to
the protein kinase inhibitor staurosporine (STS) (300 nM).
The time course of mitochondrial membrane potential (  m) during apoptosis was examined using the
probe tetramethylrhodamine ethyl ester (TMRE). Cells exhibited no
decrease in TMRE fluorescence, indicative of mitochondrial
depolarization, up to 8 hr after STS exposure. Rather, baseline TMRE
fluorescence remained unchanged up to 2 hr and thereafter actually
increased significantly. Throughout this time period, the mitochondria
could also be depolarized with the protonophore carbonyl cyanide
p-trifluoromethoxy-phenylhydrazone (FCCP, 0.1 µM), exhibiting the same relative magnitude of
fluorescence release (unquenching) as controls. Even after 16 hr of
staurosporine treatment, neurons that showed signs of nuclear apoptosis
maintained m and could be depolarized with FCCP. In
contrast, caspase-3-like activity had increased roughly sevenfold by 2 hr and >20-fold by 8 hr. Double-labeling of hippocampal neurons with
the potential-sensitive probe Mitotracker Red Chloromethyl X-Rosamine
and an antibody to cytochrome c demonstrated at the subcellular level
that mitochondrial cytochrome c release also occurred in the absence of
mitochondrial depolarization. These data suggest that mitochondrial
depolarization is not a decisive event in neuronal apoptosis.
Key words:
programmed cell death; mitochondrial membrane potential
(m); cytochrome c; permeability
transition pore (PTP); caspases; staurosporine (STS); tetramethylrhodamine ethyl ester (TMRE); carbonyl cyanide
p-trifluoromethoxy-phenylhydrazone (FCCP)
Copyright © 1999 Society for Neuroscience 0270-6474/99/19177394-11$05.00/0
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