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Mitochondrial free calcium levels (Rhod‐2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide‐dependent cell death
Author(s) -
Muriel MariePaule,
Lambeng Nathalie,
Darios Frédéric,
Michel Patrick P.,
Hirsch Etienne C.,
Agid Yves,
Ruberg Merle
Publication year - 2000
Publication title -
journal of comparative neurology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.855
H-Index - 209
eISSN - 1096-9861
pISSN - 0021-9967
DOI - 10.1002/1096-9861(20001016)426:2<297::aid-cne10>3.0.co;2-o
Subject(s) - endoplasmic reticulum , ceramide , biology , microbiology and biotechnology , programmed cell death , mitochondrion , apoptosis , biochemistry
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. J. Comp. Neurol. 426:297–315, 2000. © 2000 Wiley‐Liss, Inc.