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

Abstract

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 -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 -calpain. The immunoreactivity of activated -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.

The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia–ischemia

“…This may indicate sustained calpain activation resulting in lysosomal rupture, causing neuronal necrosis. 68 Nitric oxide (NO) is produced by three isoforms of nitric oxide synthase (NOS). Inducible and neuronal NOS (iNOS and nNOS, respectively) activities have been demonstrated to increase in models of HI in the immature brain.…”

Zinc and 4-Hydroxy-2-Nonenal Mediate Lysosomal Membrane Permeabilization Induced by H2O2in Cultured Hippocampal Neurons

“…The first two mechanisms have been extensively investigated in the nervous system, whereas the third has not been analyzed in detail in neurons. One report showed that lysosomal damage occurs in postischemic CA1 neurons (Yamashima et al, 2003).…”

“…Although lysosomal disruption has been demonstrated in primate CA1 neurons undergoing ischemic necrosis (Yamashima et al, 2003), the possible role of zinc and HNE in LMP of central neurons has not been studied in vivo. Hence, another new and significant contribution of the present study is the demonstration of the formation of zinc-containing vesicles in hippocampal neurons in vivo after kainate seizures.…”

Caspase-independent pathways of hair cell death induced by kanamycin in vivo

“…18,19 Calpains, cytosolic calcium-activated neutral cysteine proteases, are, at least in part, also associated with lysosomes. 19,20 Under appropriate stimuli, their active form dissociates from its inhibitor, calpastatin, and translocates to the cytoplasmic membrane where cytoskeletal attachments would be cleaved. 21 Active calpains participate in the regulation of both apoptosis and necrosis.…”