Regulation of a novel pathway for cell death by lysosomal aspartic and cysteine proteinases

Neuroscience. 1999;91(1):233-49. doi: 10.1016/s0306-4522(98)00566-1.


PC12 cells undergo apoptosis when cultured under conditions of serum deprivation. In this situation, the activity of caspase-3-like proteinases was elevated, and the survival rate could be maintained by treatment with acetyl-DEVD-cho, a specific inhibitor of caspase-3. In a culture of PC12 cells treated with acetyl-DEVD-cho, where caspase-3-like proteinases are not activated, CA074, a specific inhibitor of cathepsin B induced active death of the cells. Cathepsin B antisense oligonucleotides showed a similar effect to CA074 on the induction of active cell death. By double staining of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and activated caspase-3, the dying cells treated with CA074 were positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining but negative for activated caspase-3. Ultrastructurally, the cells were relatively large and had nuclei with chromatin condensation. The initiation of cell death by CA074 or the cathepsin B antisense were inhibited by the addition of pepstatin A, a lysosomal aspartic proteinase inhibitor, or by cathepsin D antisense. To examine whether this cell death pathway was present in cell types other than PC12 cells, we analysed dorsal root ganglion neurons obtained from rat embryos on the 15th gestational day, a time when they require nerve growth factor for survival and differentiation in culture. When cultured in the absence of nerve growth factor, the neurons survived in the presence of acetyl-DEVD-cho or acetyl-YVAD-cho. Under these conditions, CA074 reduced the survival rate of the neurons, which was subsequently restored by the further addition of pepstain A. These results suggest that a novel pathway for initiating cell death exists which is regulated by lysosomal cathepsins, and in which cathepsin D acts as a death factor. We speculate that this death-inducing activity is normally suppressed by cathepsin B.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aspartic Acid Endopeptidases / physiology*
  • Caspase 3
  • Caspases / metabolism
  • Cathepsin B / biosynthesis
  • Cathepsin B / genetics
  • Cathepsin D / biosynthesis
  • Cathepsin D / genetics
  • Cell Death / physiology
  • Cell Nucleus / drug effects
  • Cell Nucleus / ultrastructure
  • Cysteine Endopeptidases / physiology*
  • Cysteine Proteinase Inhibitors / pharmacology
  • Enzyme Precursors / metabolism
  • In Situ Nick-End Labeling
  • Lysosomes / enzymology*
  • Microscopy, Electron
  • Nerve Growth Factors / pharmacology
  • Oligonucleotides, Antisense / pharmacology
  • PC12 Cells
  • Protease Inhibitors / pharmacology
  • Rats


  • Cysteine Proteinase Inhibitors
  • Enzyme Precursors
  • Nerve Growth Factors
  • Oligonucleotides, Antisense
  • Protease Inhibitors
  • Casp3 protein, rat
  • Caspase 3
  • Caspases
  • Cysteine Endopeptidases
  • Cathepsin B
  • Aspartic Acid Endopeptidases
  • Cathepsin D