Detection of activated Caspase-3 (CPP32) in the vertebrate nervous system during development by a cleavage site-directed antiserum

Urase, Koko; Fujita, Eriko; Miho, Yasuko; Kouroku, Yoriko; Mukasa, Takeshi; Yagi, Yuzou; Momoi, Mariko Y.; Momoi, Takashi

doi:10.1016/s0165-3806(98)00124-2

Cited by 50 publications

(26 citation statements)

References 45 publications

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“…We used rabbit anti-activated -calpain antibodies made independently by Saido (1:300 -1,000 dilution) (1992) or ImajohOhmi (1:300 dilution) (Kikuchi, Imajoh-Ohmi, 1995), rabbit anti-LAMP-1 antibody (5 g/ml) (Furuno et al, 1989), rabbit antiactivated caspase-3 (p20/17) antibody (1:200 dilution) (Urase et al, 1998), rabbit anti-caspase-3 antibody (1:2,000, 65906E, Pharmingen), goat anti-CAD polyclonal antibody (1:200 -1,000 dilution; CPAN (N-19), Santa Cruz Biotechnology, Inc., CA), and goat anti-DFF-40 (CAD) polyclonal antibody (1:200 -1,000 dilution; CPAN (C-19), Santa Cruz Biotechnology, CA). As the original source of the immunogens for these antibodies was all human, these antibodies cross-reacted well with the monkey antigens.…”

Section: Antibodiesmentioning

confidence: 99%

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

et al. 2003

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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.

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Section: Antibodiesmentioning

confidence: 99%

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

et al. 2003

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“…In the mouse brain, activated caspase-3 was associated with apoptosis of neuroepithelial cells in early development, but not with cell death of postmitotic neurons during later development. 33 Death of cerebellar granule cells during the second week of postnatal life has been shown to be both p53-dependent and p53-independent, depending on the stimulus. 34 Expression of mRNA for the apoptosis-inhibiting protein Bcl-x has been shown to be low at birth, followed by an increase to higher levels in the adult brain.…”

Section: Other Regionsmentioning

confidence: 99%

Qualitative and quantitative estimates of apoptosis from birth to senescence in the rat brain

White

Barone

2001

Cell Death Differ

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Apoptosis is crucial for proper development of the CNS, wherein a significant percentage of all central neurons produced during early ontogeny die by apoptosis. To characterize the pattern of developmental programmed cell death, we assayed rat brainstem, neocortex, hippocampus, and cerebellum from birth through senescence. Quantitatively, using an ELISA for oligonucleosomal DNA fragments, we demonstrated that PND1 brainstem, neocortex, and hippocampus have the highest levels of fragmented DNA compared to older ages. Cerebellum displayed a large peak at PND10 and a smaller peak at PND21. Low levels were observed throughout adulthood and into senescence, which was corroborated qualitatively by agarose gel and TUNEL data. These data provide a temporal and regional baseline for further studies of the effects of perturbations of cell death during neural development. Quantitative and qualitative changes in these regional profiles of apoptosis due to environmental insults during early ontogeny may alter neuron number and function later in life. Cell Death and Differentiation (2001) 8, 345 ± 356.

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“…7,8,19 Cell pellets were lysed in PBS containing 0.2% Triton X-100 on ice for 10 min. After centrifugation at 10 0006g for 10 min, the cell extracts (30 mg protein) were subjected to SDS gel (12%) electrophoresis.…”

Section: Immunoblot Analysis Of Caspase-3 Cleavagementioning

confidence: 99%

bFGF inhibits the activation of caspase-3 and apoptosis of P19 embryonal carcinoma cells during neuronal differentiation

et al. 1999

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P19 embryonal carcinoma (EC) cells undergo apoptosis during neuronal differentiation induced by all-trans retinoic acid (RA). Caspase-3-like proteases are activated and involved in the apoptosis of P19 EC cells during neuronal differentiation. 1 Recently it has been shown that growth factor signals protect against apoptosis by phosphorylation of Bad. Phosphorylated Bad, an apoptotic member of the Bcl-2 family, cannot bind to Bcl-x L and results in Bcl-x L homodimer formation and subsequent antiapoptotic activity. In the present study, we demonstrate that this system is used generally to protect against apoptosis during neuronal differentiation. Bcl-x L inhibited the activation of caspase-3-like proteases. Basic fibroblast growth factor (bFGF) inhibited more than 90% of the caspase-3-like activity, inhibited processing of caspase-3 into its active form, and inhibited DNA fragmentation. bFGF activated phosphatidyl-inositol-3-kinase (PI3K) and stimulated the phosphorylation of Bad. Phosphorylation was inhibited by wortmannin, an inhibitor of PI3K and its downstream target Akt. Thus, Bad is a target of the FGF receptor-mediated signals involved in the protection against activation of caspase-3.

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Detection of activated Caspase-3 (CPP32) in the vertebrate nervous system during development by a cleavage site-directed antiserum

Cited by 50 publications

References 45 publications

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

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

Qualitative and quantitative estimates of apoptosis from birth to senescence in the rat brain

bFGF inhibits the activation of caspase-3 and apoptosis of P19 embryonal carcinoma cells during neuronal differentiation

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