Research ReportSerum-withdrawal-dependent apoptosis of hippocampal neuroblasts involves Ca++ release by endoplasmic reticulum and caspase-12 activation
Introduction
Numerous studies have definitively indicated that the alteration of intracellular Ca++ homeostasis plays a central role in initiating the apoptotic response of many different cell types (Orrenius et al., 2003). Recently the existence of a novel apoptotic pathway has been demonstrated, in which accumulation of misfolded proteins and perturbation of Ca++ homeostasis in the endoplasmic reticulum (ER) elicit a stress response that can lead to apoptosis through the activation of a particular caspase, the caspase-12, primarily associated with the ER membranes (Nakagawa et al., 2000, Nakagawa and Yuan, 2000). Caspase-12 has been shown to be activated by cytotoxic agents, ischemic insult, viral infection (Lamkanfi et al., 2004, Oubrahim et al., 2002, Terai et al., 2005) and in models of neurodegenerative diseases (Larner et al., 2004), hypoxic insult (Shibata et al., 2003, Mouw et al., 2003), amyotrophic lateral sclerosis (Wootz et al., 2004), prion diseases (Hetz et al., 2003), gangliosidoses (Tessitore et al., 2004) and neurodegenerative diseases with polyglutamine repeats (Kouroku et al., 2002).
On the contrary, the contribution of the ER stress and caspase-12 activation to apoptosis induced by growth factor deprivation is controversial. For example, no activation of caspase-12 after NGF withdrawal has been found in PC-12 cells (Nakagawa et al., 2000), while serum deprivation leads to processing of caspase-12 in retinal cells (Gomez-Vicente et al., 2005) and in AKR-2B fibroblasts (Kilic et al., 2002, Hoppe et al., 2002, Hoppe and Hoppe, 2004).
Consequently the contribution of the ER stress to apoptosis induced by serum deprivation is still unclear. In the present paper, we have analyzed the cellular response of immortalized embryonic hippocampal neuroblasts to serum withdrawal to clarify the involvement of the ER-dependent pathway to neuronal apoptosis triggered by serum deprivation. We have previously shown that serum deprivation increases ceramide levels, produces relocation of cytochrome c to cytosol and of Bax to mitochondria, followed by deregulation of calcium metabolism without loss of mitochondrial functionality (Colombaioni et al., 2002a) as well as activation of caspase-3 (Colombaioni et al., 2002b) in accord with other neuronal models of serum deprivation (Mukasa et al., 1997). With this study we provide evidence that serum deprivation promotes the overexpression of the protein GRP-78, a well-known marker of ER stress, depletion of ER Ca++ and processing of caspase-12 earlier than caspase-3 activation.
Section snippets
Serum deprivation induces apoptosis of hippocampal neuroblasts
Immortalized hippocampal neuroblasts were serum deprived by lowering fetal bovine serum (FBS) content in the culture medium from 10% to 0.2%. Apoptosis incidence was evaluated, at various time points, by staining cells with the DNA-binding dye Hoechst 33258. Apoptotic cells were recognized on the basis of the typical morphology of nucleus that appeared condensed and fragmented (Fig. 1A). No significant increase of the apoptosis incidence was detected during the first 4 h of treatment; however,
Discussion
With this work we have investigated the involvement of ER stress in the initiation of apoptosis elicited by serum deprivation in undifferentiated hippocampal neuroblasts. We have found that serum deprivation transiently enhances the expression of GRP-78, a marker of ER stress. This event is followed by a coordinated decrease of [Ca++]ER and increase of [Ca++]MTC, followed by the cleavage of procaspase-12. The direct [Ca++]ER depletion by thapsigargin, a specific inhibitor of the ER-associated Ca
Cell culture and apoptosis evaluation
Immortalized neuroblasts from mouse hippocampus (cell line HN9.10e) were cultured in F-12 (Sigma) containing 10% FBS, at 37 °C under 5% CO2. Cell death was assessed on the basis of cellular and nuclear morphology observed by fluorescence microscopy after staining with 2 μM Hoechst 33258. At least 500 cells were counted for each treatment, and the data were expressed as the mean ± SEM from at least 3 independent experiments. P values were calculated by t-test and regarded as significant when P
Acknowledgments
This work was funded by the University of Pisa and by the Consiglio Nazionale delle Ricerche.
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