Elsevier

Brain Research

Volume 1147, 25 May 2007, Pages 1-11
Brain Research

Research Report
Serum-withdrawal-dependent apoptosis of hippocampal neuroblasts involves Ca++ release by endoplasmic reticulum and caspase-12 activation

https://doi.org/10.1016/j.brainres.2007.01.145Get rights and content

Abstract

Apoptotic death caused by diseases or toxic insults is preceded and determined by endoplasmic reticulum dysfunction and altered intraluminar calcium homeostasis in many different cell types. With the present study we have explored the possibility that the ER stress could be involved also in apoptotic death induced by serum deprivation in neuronal cells. We have chosen as a model of study the cell line HN9.10e, constituted by immortalized hippocampal neuroblasts. The Ca++ concentration in the lumen of the ER has been evaluated by using the low affinity Ca++ probe Mag-fluo-4. We show that serum deprivation lowers the ER Ca++ concentration with a time course closely related to the increase of apoptosis incidence. Serum deprivation also enhances the expression of a well-known marker of ER stress, the glucose-regulated protein-78 (GRP-78), a member of the heat shock/stress response protein family. Moreover, in serum-deprived neuroblasts, following GRP-78 up-regulation, the ER-associated procaspase-12 is cleaved with a time course which parallels the ER calcium loss while activation of caspase-3 is a later event. Depletion of ER Ca++ by thapsigargin, a specific inhibitor of the ER-associated Ca++ ATPase, also produces caspase-12 processing and apoptotic cell death, whereas agents capable of reducing the ER calcium loss protect the cells from serum-deprivation-induced apoptosis. These findings indicate that, in hippocampal neuroblasts, Ca++ mobilization from ER and caspase-12 activation are components of the molecular pathway that leads to apoptosis triggered by serum deprivation and may constitute an amplifying loop of the mitochondrial pathway.

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.

References (42)

  • Y. Tan et al.

    Ubiquitous calpains promote caspase-12 and JNK activation during endoplasmic reticulum stress-induced apoptosis

    J. Biol. Chem.

    (2006)
  • A. Tessitore et al.

    GM1-ganglioside-mediated activation of the unfolded protein response causes neuronal death in a neurodegenerative gangliosidosis

    Mol. Cell

    (2004)
  • H. Wootz et al.

    Caspase-12 cleavage and increased oxidative stress during motoneuron degeneration in transgenic mouse model of ALS

    Biochem. Biophys. Res. Commun.

    (2004)
  • Z. Yu et al.

    The endoplasmic reticulum stress-responsive protein GRP78 protects neurons against excitotoxicity and apoptosis: suppression of oxidative stress and stabilization of calcium homeostasis

    Exp. Neurol.

    (1999)
  • D.F. Babcock et al.

    Mitochondrial participation in the intracellular Ca2+ network

    J. Cell Biol.

    (1997)
  • D. Boehning et al.

    Cytochrome c binds to inositol (1,4,5) triphosphate receptors, amplifying calcium-dependent apoptosis

    Nat. Cell Biol.

    (2003)
  • D.G. Breckenridge et al.

    Regulation of apoptosis by endoplasmic reticulum pathways

    Oncogene

    (2003)
  • D.G. Breckenridge et al.

    Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol

    J. Cell Biol.

    (2003)
  • D. Chandra et al.

    Association of active caspase 8 with the mitochondrial membrane during apoptosis: potential roles in cleaving BAP31 and caspase 3 and mediating mitochondrion-endoplasmic reticulum cross talk in etoposide-induced cell death

    Mol. Cell. Biol.

    (2004)
  • F. Darios et al.

    Ceramide increases mitochondrial free calcium levels via caspase 8 and Bid: role in initiation of cell death

    J. Neurochem.

    (2003)
  • L. Dreier et al.

    In vitro formation of the endoplasmic reticulum occurs independently of microtubules by a controlled fusion reaction

    J. Cell Biol.

    (2000)
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