Cell death as a result of exposure to ionising radiation has been extensively investigated; however, the complexity of the various mechanisms involved in this response remains a key topic of interest. DNA is known to be the most critical radio-sensitive component of cells, directly targeted by radiation or indirectly via water radiolysis that produces reactive oxygen species. These products are responsible for the induction of damage to DNA, including double-strand breaks (24
), the most damaging lesion that can lead, in the case of repair failure, to cell death, particularly following exposure to low doses of ionising radiation (26
). Double-strand breaks were revealed in this study by detecting the phosphorylated histone, H2AX (γ-H2AX), as one of the most effective markers of response to radiation-induced double-strand breaks. This response triggers a signaling cascade by the activation of an important component in double-strand break signaling, the ATM protein kinase. ATM is responsible for the phosphorylation of the H2AX histone and the indirect activation of cell cycle check-points proteins required for cell cycle arrest and DNA repair (27
). ATM also regulates the P53 protein, known as the guardian of the genome (28
) for its key role in stress response by the induction of cell cycle arrest, DNA repair and apoptosis regulation (29
). Its activation has been widely associated with cell death induction (30
), a phenomenon that was observed in this study following exposure to the moderate doses of 0.2 and 0.5 Gy.
Nevertheless, in the nervous system, multiple pathways leading to neuronal death exist depending on the nature of the stressor, and involve key proteins, such as the Bcl-2 family responsible for the induction of the mitochondrial pathway, leading to the activation of caspase proteins (32
) and calpains, calcium-dependent enzymes, involved in cell death induction (33
). Evidence of a crosstalk between these pathways makes the process even more complex. A particularity of the neuronal system is the excitability of the cells. The over-activation of NMDA receptors by a high concentration of glutamate, the main excitatory neurotransmitter in the mammalian CNS, causes the cells to die from excitotoxicity, due to a massive entry of calcium ions inside the cell (34
). NMDArs are glutamate-gated ion channels, which are selectively activated by the artificial glutamate analog, NMDAr. These channels when open, are highly permeable to Ca2+
Attention has been paid to the pathological significance of calcium accumulation in the CNS following insult to the brain, including radiation damage. Excitotoxicity is linked to chronic neurological disorders, including Alzheimer’s and Parkinson’s disease (17
), and acute CNS insults, including hypoxia/ischemia (36
). Over-activation of NMDArs in the brain leads to a sustained influx of Ca2+
through NMDA and non-NMDA Ca2+
channels. Such disturbances in calcium homeostasis may result in the activation of several calcium-dependent cysteine proteases, including calpain (an intracellular cysteine protease proenzyme activated by autocatalytic cleavage in the presence of high calcium concentrations) and caspases involved in cytotoxicity downstream (37
). Hence, the selective inhibition of calcium entry by the blockade of ion gated channels to limit neuronal damage after irradiation appears to be an attractive method of evaluating the role of calcium homeostasis in the radiation-induced neurodegenerative processes. We therefore investigated the possible role of NMDAr and Ca2+
in the induction of radiation-induced neuronal cell apoptosis.
We showed that a 0.6 Gy of X-ray exposure in utero, led to a clear apoptotic response in E15 fetal rat cortices. This apoptotic response was not observed in the different fetal brains of non-irradiated animals used as the controls (Sham-exposed and MK-801, nimodipine or calpain inhibitor-treated animals). The same results were obtained following irradiation of 7-day primary cultures of cortical neurons with 0.2 and 0.5 Gy using the TUNEL test, which indicated radiation-induced cell death. Caspase-3 activity, a key factor in apoptosis induction, was also increased following exposure to the same doses indicating that cell death by apoptosis is caspase-dependent. However, following irradiation, the cell death index was higher than caspase-3 activity, suggesting that other apoptotic mechanisms which are caspase-3-independent may be responsible for this difference in response to radiation. The number of TUNEL-stained cells and caspase-3 activity were not significantly increased in the control cultures (non-irradiated but treated with MK-801 or calpain inhibitor) and the cultures irradiated with the low dose of X-rays.
The apoptotic response including DNA fragmentation (TUNEL) and caspase-3 activation induced in the irradiated cultures with 0.2 and 0.5 Gy was prevented by treatment with MK-801, which selectively blocks NMDAr and neuronal Ca2+ influx. This indicates that radiation-induced apoptosis is mediated through NMDAr and is affected by massive entry of Ca2+ into the cells.
Calpain was also a good candidate in excitotoxicity-mediated neuronal death; thus, neuronal cultures were treated with calpain inhibitor prior to irradiation. Our results showed that calpain inhibitor prevented the apoptotic response in irradiated cultures, thus supporting our hypothesis of the importance of a calpain-mediated effect in radiation-induced apoptosis in the fetal brain.
Similar results were also observed after in vivo treatment of pregnant rats by an injection of MK-801 or calpain inhibitor 20 min following exposure to 0.6 Gy of X-rays. Both treatments prevented DNA laddering, indicating that they can protect the fetal brain from apoptotic response. The in vivo experiment also allowed us to eliminate the implication of other Ca2+ channels in this radiation-induced excitotoxicity, such as the L-type, high threshold and voltage-dependent Ca2+ channels. The blockade of these channels by nimodipine did not prevent irradiation-induced DNA laddering; Therefore, it cannot protect the fetal brain from radiation-induced apoptosis, indicating that the sensitivity of the fetal brain to Ca2+ influx through NMDA channels is specific and indicates a particular radiosensitivity of the cell bearing these receptors. Thus, apoptosis induced in immature neurons, by activation of Ca2+-dependent proteolytic enzymes such as calpain, plays a key role in the radiation-induced damage of the developing fetal brain.
Our results showing the protective effect of either MK-801 or calpain inhibitor on radiation-induced apoptosis in the fetal cortex and in vitro, specifically in established neuronal network of 7-day cultured cortical neurons, further suggest the involvement of various pathways leading to neuronal cell death following exposure to low and moderate doses of ionising radiation.
Indeed, the activation of caspase-3 that was observed following irradiation is a classical response to Ca2+
influx, responsible for apoptosis induction by the cleavage of several proteins involved in this process. The inhibition of caspase-3 protects cortical neurons from NMDAr-induced apoptosis (38
). The activation of caspase-3 has been reported to be a downstream effector of mitochondrial disruption following the release of cytochrome c (38
) and is involved in the execution phase of apoptosis.
On the other hand, calpain is involved in several actions following the entry of calcium. Calpain is a proteolytic enzyme directly activated by calcium entry (40
) and is mainly known for its capacity to cleave cytoskeletal proteins, such as α-spectrin, a phenomenon that suggests its important role in various neurodegenerative diseases (41
). Attention has been paid to the novel roles of calpain in the excitotoxicity phenomenon. It has been found to contribute to the further disturbance of calcium homeostasis by cleaving different substrates involved in calcium extruding, such as the Na+
exchanger and sarcoplasmic/endoplasmic reticulum calcium ATPase (42
) or in cytosolic calcium homeostasis, such as the protein phosphatase calcineurin (44
). When activated following the cleavage by calpain, the latter triggers downstream effectors known to induce apoptosis, including cytochrome c release from the mitochondria, leading to caspase-3 activation. This has been further proven by the overexpression of 48-kDa calcineurin A (truncated active form), that has been shown to induce an increase in caspase-3 activity and TUNEL-positive apoptotic cells (44
). The same finding has been reported using a Parkinson’s disease model, where caspase-3 activation was calpain-dependent (45
). A recent study also established a link between the calcium-dependent activation of calpain and the induction of apoptosis via caspases-12, 9 and 3 (46
). Our results showing a decrease in caspase-3 activity and DNA fragmentation following treatment with calpain inhibitor also confirm these findings, which permit us to establish a link between calpain and caspase-3 activity, a link that has not always been clear since these two enzymes were believed to be involved in two independent pathways ultimately leading to cell death. Other studies had even described caspase-3 as being directly activated following cleavage by calpain (47
), indicating another contribution of calpain to the apoptotic induction of caspase-dependent apoptosis.
Our results also demonstrate a radiation-induced DNA damage by detecting double-strand breaks. This damage was shown to proportionally increase with the dose. Such damage is believed to enhance the expression of P53 protein which plays a key role in apoptosis induction (49
) through the activation of Bax, a pro-apoptotic protein (50
). A P53-dependent activation of Bax has also been shown to be involved in NMDAr-mediated neuronal death (51
). Of note, it has been found that calpain activity may be induced following DNA damage and furthermore leads to the activation of P53 (52
), indicating another role of calpain in the induction of caspase-dependent apoptosis via the activation of P53 response following DNA damage. Furthermore, the fact that the inhibition of calpain in our study almost completely prevented cells from radiation induced-apoptosis, including the fraction of cells that died independently from caspase-3 activation, leads us to suggest an involvement of calpain in both caspase-dependent and -independent pathways.
These studies together with our results indicate a central role of calpain in radiation-induced excitotoxicity, but also indicate an evident crosstalk of several cell death pathways. These interactions and their nature (synergistic or competitive), remain poorly understood; thus investigating these interactions is of high interest for the elaboration of neuroprotective therapies for neurodegenerative diseases caused by excitotoxicity and this study opens new perspectives for radiation protection of the developing brain.
Our results reveal a new non-conventional radiation-induced cell death pathway, involving the excitotoxicity principle mediated by NMDAr activation, not dependent on direct radiation DNA damage. This pathway involves the activation of calpain enzyme but also caspase-3 activation, suggesting the eventual direct or indirect interaction of these two proteins and their respective classical pathways. P53 activation by calpain following radiation-induced DNA damage remains a hypothesis that requires further investigation.