Sustained calpain activation associated with lysosomal rupture executes necrosis of the postischemic CA1 neurons in primates

Hippocampus. 2003;13(7):791-800. doi: 10.1002/hipo.10127.

Abstract

Because of the paucity of primate experimental models, the precise molecular mechanism of ischemic neuronal death remains unknown in humans. This study focused on nonhuman primates to determine which cascade necrosis or apoptosis is predominantly involved in the development of delayed (day 5) neuronal death in the hippocampal CA1 sector undergoing 20 min ischemia. We investigated expression, activation, and/or translocation of micro-calpain, lysosome-associated membrane protein-1 (LAMP-1), caspase-3, and caspase-activated DNase (CAD), as well as morphology of the postischemic CA1 neurons and DNA electrophoresis pattern. Immunoblotting showed sustained (immediately after ischemia until day 5) and maximal (day 3) activation of micro-calpain. The immunoreactivity of activated micro-calpain became remarkable as coarse granules at lysosomes on day 2, while it translocated throughout the perikarya on day 3. The immunoreactivity of LAMP-1 also showed a dynamic and concomitant translocation that was maximal on days 2-3, indicating calpain-mediated disruption of the lysosomal membrane after ischemia. In contrast, immunoblotting demonstrated essentially no increase in the activated caspase-3 at any time points after ischemia, despite upregulation of pro-caspase-3. Although expression of CAD was slightly upregulated on day 1 or 2, or both, it was much less compared with lymph node or intestine tissues. Furthermore, light and electron microscopy showed eosinophilic coagulation necrosis and membrane disruption without apoptotic body formation, while DNA electrophoresis did not show a ladder pattern, but rather a smear pattern. Sustained calpain activation and the resultant lysosomal rupture, rather than CAD-mediated apoptosis, may cause ischemic neuronal necrosis in primates.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, CD / metabolism
  • Brain Ischemia / enzymology*
  • Brain Ischemia / pathology
  • Brain Ischemia / physiopathology
  • Calpain / metabolism*
  • Caspase 3
  • Caspases / metabolism
  • Deoxyribonucleases / metabolism
  • Disease Models, Animal
  • Hippocampus / enzymology
  • Hippocampus / pathology
  • Hippocampus / physiopathology
  • Immunohistochemistry
  • Lysosome-Associated Membrane Glycoproteins
  • Lysosomes / enzymology*
  • Lysosomes / ultrastructure
  • Macaca / metabolism*
  • Microscopy, Electron
  • Necrosis
  • Neurons / enzymology*
  • Neurons / pathology
  • Neurons / ultrastructure
  • Protein Transport / physiology
  • Reaction Time / physiology
  • Up-Regulation / drug effects
  • Up-Regulation / physiology

Substances

  • Antigens, CD
  • Lysosome-Associated Membrane Glycoproteins
  • Deoxyribonucleases
  • caspase-activated deoxyribonuclease
  • Calpain
  • Caspase 3
  • Caspases
  • mu-calpain