To explore how the intrinsic apoptosis pathway is controlled in the spontaneous fog （forebrain overgrowth） mutant mice with an Apafl splicing deficiency, we examined spleen and bone marrow cells from Apafl＋/＋ （＋...To explore how the intrinsic apoptosis pathway is controlled in the spontaneous fog （forebrain overgrowth） mutant mice with an Apafl splicing deficiency, we examined spleen and bone marrow cells from Apafl＋/＋ （＋/＋） and Apafl^fog/fog （fog/fog） mice for initiator caspase-9 activation by cellular stresses. When the mitochondrial inner membrane potential （△ψm） was disrupted by staurosporine, ＋/＋ cells but not fog/fog cells activated caspase-9 to cause apoptosis, indicating the lack of apoptosome （apoptosis protease activating factor 1 （Apaf-l）/cytochrome c/（d）ATP/procaspase-9） function in fog/fog cells. However, when a marginal （-20%） decrease in △ψm was caused by hydrogen peroxide （0.1 mM）, peroxynitrite donor 3-morpholinosydnonimine （0.1 mM） and UV-C irradiation （20 J/m^2）, both ＋/＋ and fog/fog cells triggered procaspase-9 auto-processing and its downstream cascade activation. Supporting our previous results, procaspase-9 pre-existing in the mitochondria induced its auto-processing before the cytosolic caspase activation regardless of the genotypes. Cellular ATP concentration significantly decreased under the hypoactive △ψm condition. Furthermore, we detected accumulation of citrate, a kosmotrope known to facilitate procaspase-9 dimerization, probably due to a feedback control of the Krebs cycle by the electron transfer system. Thus, mitochondrial in situ caspase-9 activation may be caused by the major metabolic reactions in response to physiological stresses, which may represent a mode of Apaf-l-independent apoptosis hypothesized from recent genetic studies.展开更多
Previous investigations of retrograde survival signaling by nerve growth factor （NGF） and other neurotrophins have supported diverse mechanisms, but all proposed mechanisms have in common the generation of survival ...Previous investigations of retrograde survival signaling by nerve growth factor （NGF） and other neurotrophins have supported diverse mechanisms, but all proposed mechanisms have in common the generation of survival signals retrogradely transmitted to the neuronal cell bodies. We report the finding of a retrograde apoptotic signal in axons that is suppressed by local NGF signaling. NGF withdrawal from distal axons alone was sufficient to activate the pro-apoptotic transcription factor, c-jun, in the cell bodies. Providing NGF directly to cell bodies, thereby restoring a source of NGF-induced survival signals, could not prevent c-jun activation caused by NGF withdrawal from the distal axons. This is evidence that c-jun is not activated due to loss of survival signals at the cell bodies. Moreover, blocking axonal transport with colchicine inhibited c-jun activation caused by NGF deprivation suggesting that a retrogradely transported pro-apoptotic signal, rather than loss of a retrogradely transported survival signal, caused c-jun activation. Additional experiments showed that activation of c-jun, pro-caspase-3 cleavage, and apoptosis were blocked by the protein kinase C inhibitors, rottlerin and chelerythrine, only when applied to distal axons suggesting that they block the axon-specific pro-apoptotic signal. The rottlerin-sensitive mechanism was found to regulate glyco- gen synthase kinase 3 （GSK3） activity. The effect of siRNA knockdown, and pharmacological inhibition of GSK3 suggests that GSK3 is required for apoptosis caused by NGF deprivation and may function as a retrograde carrier of the axon apoptotic signal. The existence of a retrograde death signaling system in axons that is suppressed by neurotro- phins has broad implications for neurodevelopment and for discovering treatments for neurodegenerative diseases and neurotrauma.展开更多
文摘To explore how the intrinsic apoptosis pathway is controlled in the spontaneous fog （forebrain overgrowth） mutant mice with an Apafl splicing deficiency, we examined spleen and bone marrow cells from Apafl＋/＋ （＋/＋） and Apafl^fog/fog （fog/fog） mice for initiator caspase-9 activation by cellular stresses. When the mitochondrial inner membrane potential （△ψm） was disrupted by staurosporine, ＋/＋ cells but not fog/fog cells activated caspase-9 to cause apoptosis, indicating the lack of apoptosome （apoptosis protease activating factor 1 （Apaf-l）/cytochrome c/（d）ATP/procaspase-9） function in fog/fog cells. However, when a marginal （-20%） decrease in △ψm was caused by hydrogen peroxide （0.1 mM）, peroxynitrite donor 3-morpholinosydnonimine （0.1 mM） and UV-C irradiation （20 J/m^2）, both ＋/＋ and fog/fog cells triggered procaspase-9 auto-processing and its downstream cascade activation. Supporting our previous results, procaspase-9 pre-existing in the mitochondria induced its auto-processing before the cytosolic caspase activation regardless of the genotypes. Cellular ATP concentration significantly decreased under the hypoactive △ψm condition. Furthermore, we detected accumulation of citrate, a kosmotrope known to facilitate procaspase-9 dimerization, probably due to a feedback control of the Krebs cycle by the electron transfer system. Thus, mitochondrial in situ caspase-9 activation may be caused by the major metabolic reactions in response to physiological stresses, which may represent a mode of Apaf-l-independent apoptosis hypothesized from recent genetic studies.
文摘Previous investigations of retrograde survival signaling by nerve growth factor （NGF） and other neurotrophins have supported diverse mechanisms, but all proposed mechanisms have in common the generation of survival signals retrogradely transmitted to the neuronal cell bodies. We report the finding of a retrograde apoptotic signal in axons that is suppressed by local NGF signaling. NGF withdrawal from distal axons alone was sufficient to activate the pro-apoptotic transcription factor, c-jun, in the cell bodies. Providing NGF directly to cell bodies, thereby restoring a source of NGF-induced survival signals, could not prevent c-jun activation caused by NGF withdrawal from the distal axons. This is evidence that c-jun is not activated due to loss of survival signals at the cell bodies. Moreover, blocking axonal transport with colchicine inhibited c-jun activation caused by NGF deprivation suggesting that a retrogradely transported pro-apoptotic signal, rather than loss of a retrogradely transported survival signal, caused c-jun activation. Additional experiments showed that activation of c-jun, pro-caspase-3 cleavage, and apoptosis were blocked by the protein kinase C inhibitors, rottlerin and chelerythrine, only when applied to distal axons suggesting that they block the axon-specific pro-apoptotic signal. The rottlerin-sensitive mechanism was found to regulate glyco- gen synthase kinase 3 （GSK3） activity. The effect of siRNA knockdown, and pharmacological inhibition of GSK3 suggests that GSK3 is required for apoptosis caused by NGF deprivation and may function as a retrograde carrier of the axon apoptotic signal. The existence of a retrograde death signaling system in axons that is suppressed by neurotro- phins has broad implications for neurodevelopment and for discovering treatments for neurodegenerative diseases and neurotrauma.
