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Published online 21 August 2000. doi:10.1083/jcb.150.4.741
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© The Rockefeller University Press, 0021-9525/2000//741 $5.00
The Journal of Cell Biology, Volume 150, Number 4, , 2000 741-754

Original Article

Death Commitment Point Is Advanced by Axotomy in Sympathetic Neurons



Graham C. Fletchera, Luzheng Xuea, Shareta K. Passinghama, and Aviva M. Tolkovskya

a Department of Biochemistry, University of Cambridge, Downing Site, Cambridge, CB2 1QW, United Kingdom
Department of Biochemistry, University of Cambridge, Building 0, Downing Site, Cambridge, CB2 1QW, UK.44-122-333-334544-122-333-9319

amt{at}mole.bio.cam.ac.uk

Axotomized neurons have several characteristics that are different from intact neurons. Here we show that, unlike established cultures, the axotomized sympathetic neurons deprived of NGF become committed to die before caspase activation, since the same proportion of NGF-deprived neurons are rescued by NGF regardless of whether caspases are inhibited by the pan-caspase inhibitor Boc-Asp(O-methyl)-CH2F (BAF). Despite prolonged Akt and ERK signaling induced by NGF after BAF treatment has prevented death, the neurons fail to increase protein synthesis, recover ATP levels, or grow. Within 3 d, all the mitochondria disappear without apparent removal of any other organelles or loss of membrane integrity. Although NGF does rescue intact BAF-treated 6-d cultures after NGF deprivation, rescue by NGF fails when these neurons are axotomized before NGF deprivation and BAF treatment. Moreover, cytosolic cytochrome c rapidly kills axotomized neurons. We propose that axotomy induces signals that make sympathetic neurons competent to die prematurely. NGF cannot repair these NGF-deprived, BAF-treated neurons because receptor signaling (which is normal) is uncoupled from protein renewal, and the mitochondria (which are damaged) go on to be eliminated. Hence, the order of steps underlying neuronal death commitment is mutable and open to regulation.

Key Words: apoptosis • caspases • mitochondria • NGF • survival signaling

© 2000 The Rockefeller University Press

Abbreviations used in this paper: BAF, t-butoxy carbonyl-Asp(O-methyl)-CH2F; CPTcAMP, 8-(4-chloropheny thio) cAMP; cyt c, cytochrome c; fmk, fluoromethylketone; MTT, 3-[4,5 dimethylthiazol-2-phenyl]-2,5-diphenyl-tetrazolium; SCG, superior cervical ganglia; zVAD.fmk, benzyloxyonyl-Val-Ala-Asp(O-methyl)-CH2F; 0-DIV, newly isolated SCG neurons; 6-DIV, 6-day cultured SCG neurons.


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