Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death.
Mitochondrial free calcium levels measured by Rhod-2 fluorescence and ultrastructure were examined during cell death in nerve growth factor (NGF)-differentiated PC12 cells that were 1) exposed to C2-ceramide, 2) deprived of serum to induce endogenous ceramide production, or 3) treated with calcium ionophore A23187. Rhod-2 fluorescence in mitochondria and also in the nucleolus increased to a maximum within 3 hours after C2-ceramide treatment or serum withdrawal. In A23187-treated cells, Rhod-2 fluorescence remained at baseline levels. In all three models, enlargement of the endoplasmic reticulum was the first ultrastructural alteration, followed by mitochondrial shrinkage in ionophore-treated cells, but by mitochondrial swelling in the ceramide-dependent models, in which rupture of the outer mitochondrial membrane and unfolding of the inner membrane were frequently seen. Dihydro-C2-ceramide, which did not cause cell death, had no effect on cellular ultrastructure. NGF, which inhibits ceramide-dependent cell death, prevented the effects of serum deprivation on mitochondrial ultrastructure but not on endoplasmic reticulum morphology or Rhod-2 fluorescence. Nuclear shrinkage with loss of nuclear membrane integrity, characterized by nuclear pores, free or surrounded by electron-dense filaments, was a late event in ceramide-dependent cell death. Chromatin condensation and other morphological features associated with apoptosis were seen in only a few atypical cells. Ceramide-mediated cell death, therefore, did not involve classical apoptosis but was mediated by a reproducible series of events beginning in the endoplasmic reticulum, followed by the mitochondria, and then the nucleus. NGF-dependent cell death inhibition intervenes at the mitochondrial level, not by blocking the increase in Rhod-2 fluorescence but by preventing the ultrastructural changes that follow.
Muriel
M P
MP
INSERM U289, Hôpital de la Salpêtrière, 75013 Paris, France.
Lambeng
N
N
Darios
F
F
Michel
P P
PP
Hirsch
E C
EC
Agid
Y
Y
Ruberg
M
M
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology; D002454:Cell Differentiation; D004721:Endoplasmic Reticulum / Q000502:physiology; D005453:Fluorescence; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D008928:Mitochondria / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure*; D009474:Neurons / Q000473:pathology*; D016716:PC12 Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013997:Time Factors
D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; C064769:N-acetylsphingosine; C068483:rhod-2; D000001:Calcimycin; D013110:Sphingosine; D002118:Calcium