Harnessing cell death

Cell death is an essential process for all multicellular organisms and its malfunction is involved in several diseases, but can we utilise it? Dr Benjamin Bonneau tells us about a new regulator of apoptosis and discusses how it could be used against cancer and Alzheimer’s disease 

Calcium is a central actor in the life of all multicellular organisms – it participates in almost every aspect of cell activity from cell division to cell death. Calcium is a versatile intracellular messenger which reaches various targets depending on the localisation and the strength of calcium signal. The endoplasmic reticulum (ER) is acknowledged to be the main intracellular calcium store as the calcium concentration within the ER is 1000 to 10,000-fold higher than in the cytosol. The generation of calcium signals originating from the ER mainly depends on the opening of the ER-resident calcium channel, inositol 1,4,5-trisphosphate receptor (IP3R)¹.

Calcium can indeed induce apoptosis by acting on mitochondria, the organelle responsible for energy production in the cell

Cellular response following IP3R opening depends on the amount of calcium released through the channel. For instance, small oscillatory calcium concentration elevation promotes cell survival while sustained robust calcium concentration elevation induces cell death. IP3R activity is then tightly regulated by various interacting partners that modulate calcium release according to cellular context². This is the case for the proteins belonging to the Bcl-2 family, a group of proteins that control the programmed cell death also known as apoptosis. Some members of the Bcl-2 family proteins promote cell survival or inhibit cell death by regulating the amount of calcium released through IP3R³. Calcium can indeed induce apoptosis by acting on mitochondria, the organelle responsible for energy production in the cell. To work, mitochondria require calcium which is constantly provided by some portions of the ER physically attached to mitochondria and known as mitochondria-associated membranes or MAMs. However, in some conditions, the amount of calcium released from the ER can dramatically increase and due to close proximity between ER and mitochondria at MAMs, the calcium is directly transferred to mitochondria leading to a so-called “mitochondrial calcium overload” which eventually triggers apoptosis⁴.

Apoptosis is a process that occurs physiologically in all multicellular organisms. This programmed cell death is in constant equilibrium with cell division and proliferation, allowing the maintenance of the number of cells in a tissue or an organism. It plays a key role in many vital processes – for example embryogenesis and immune system functioning. Several pathologies originate from the malfunction of apoptotic process; this is the case notably for cancers where cells are no longer able to die, or for neurodegenerative and autoimmune diseases where excessive apoptosis occurs⁵. The understanding of the regulatory mechanisms of apoptosis is then essential as they represent potential therapeutical targets.

Mitochondrial contact In our recent study, published in eLife, we described a new regulatory mechanism of apoptosis mediated by the protein IRBIT. This protein was originally discovered in our laboratory as a protein interacting with IP3R and decreasing the release of calcium through the channel. To interact with IP3R and exert its effect on calcium signalling, IRBIT need to be phosphorylated. In addition to IP3R, IRBIT can regulate the activity of several other partners such as ions channels or kinases⁶. In the study⁷, we found that IRBIT can also interact with Bcl2l10, a protein belonging to the Bcl-2 family and preventing apoptosis. Bcl2l10 also interacts with IP3R and decrease calcium release from the ER. Interestingly, while IRBIT needs to be phosphorylated to interact with IP3R and almost all its other partners, the interaction between Bcl2l10 and IRBIT does not require IRBIT phosphorylation. IRBIT and Bcl2l10 are in fact associated on IP3R and this complex is present in MAMs, the place in cells where calcium is transferred from the ER to the mitochondria.

We were then astonished to discover that IRBIT deletion, using CRISPR/Cas9 technique, in fact renders cells highly resistant to apoptosis meaning that IRBIT promotes cell death

Given it decreases calcium release through IP3R and interacts with Bcl2l10, a protein preventing apoptosis, we first thought that IRBIT may also have some anti-apoptotic features. We were then astonished to discover that IRBIT deletion, using CRISPR/Cas9 technique, in fact renders cells highly resistant to apoptosis meaning that IRBIT promotes cell death. From our results it appears that IRBIT favours apoptosis by two mechanisms. The first one relies on IRBIT dephosphorylation – we found that at the onset of apoptosis, IRBIT is dephosphorylated. unphosphorylated IRBIT then interacts with Bcl2l10 but not with IP3R – the dephosphorylation of IRBIT induces the translocation of both IRBIT and Bcl2l10 from MAMs. The removal of two proteins normally reducing calcium release through IP3R allows a massive calcium transfer to mitochondria which leads to mitochondrial calcium overload and then apoptosis. In addition to this mechanism, we discovered that IRBIT also increases the amount of ER in contact with mitochondria which also favours the transfer of calcium to mitochondria during apoptosis (Figure 1).

Our study then revealed a new function of IRBIT as a regulator of cell death through its ability to increase ER-mitochondria contact and to inhibit the activity of the anti-apoptotic protein Bcl2l10. This finding may have implications regarding a possible role for IRBIT in cancers and neurodegenerative diseases.

[caption id="attachment_59644" align="alignnone" width="620"] Schematic model depicting the role of IRBIT in cell death.
In physiological condition, in WT cells, IRBIT promotes ER-mitochondria contact rendering Ca2+ transfer easier between the two organelles. The additive effect of Bcl2l10 and phosphorylated IRBIT on IP3R maintains Ca2+ transfer to a low level. In IRBIT KO cells, although Ca2+ released through IP3R is increased due to absence of IRBIT, Ca2+ transfer to the mitochondria is reduced because of the great reduction of ER-mitochondria contact. Following an apoptotic stimuli, Ca2+ release from the ER is increased. In WT cells, IRBIT dephosphorylation induces its translocation together with Bcl2l10 allowing a massive Ca2+ transfer from ER to mitochondria. On contrary, in IRBIT KO cells, Bcl2l10 is no longer displaced from MAMs what reduces Ca2+ release from ER. This, combined to the reduction of ER-mitochondria contact, prevents massive Ca2+ transfer to mitochondria and then greatly attenuates apoptosis.[/caption]

Our results now suggest that reduced expression of IRBIT can, in fact, actively participate in the malignant phenotype of cancer cells

Oncological promise In cancer, cells gain the ability to proliferate quickly while they lose their ability to die. This immortality is generally provided by the altered expression of Bcl-2 family proteins; and Bcl2l10 expression was found to be increased in various cancers. In contrast, IRBIT expression was found to be reduced in several cancer cell lines but the significance of this and how it can be related to cancer formation has not been established^8,9. Our results now suggest that reduced expression of IRBIT can, in fact, actively participate in the malignant phenotype of cancer cells. Indeed, we can assume that the reduction of IRBIT expression in cancer cell leads to an increased activity of Bcl2l10, which may promote apoptosis resistance in these cells. Moreover, based on our results we can now say that decreased expression of IRBIT leads to a reduction of the contact between ER and mitochondria. As these contacts are important for apoptosis to occur, cells with reduced MAMs are more resistant to death and therefore, prone to become cancerous.

In cancer, the cells divide very quickly and high activity of RNR is then essential to supply the dNTPs necessary for rapid cell proliferation

It was also recently shown that IRBIT inhibits ribonucleotide reductase (RNR) – an enzyme providing the dNTPs, the building blocks for DNA¹⁰. During cell division, RNR activity is essential to provide the dNTP required to double the quantity of DNA. In cancer, the cells divide very quickly and high activity of RNR is then essential to supply the dNTPs necessary for rapid cell proliferation. In this respect, increased RNR activity is frequently observed in cancer. Thus, as IRBIT inhibits RNR activity, a reduced expression of IRBIT may allow an increased activity of RNR which can in turn promote cell proliferation.

Therefore, the reduction of IRBIT expression may confer apoptosis resistance but also facilitate rapid cell proliferation, two of the main hallmarks of cancer cells. We can then hypothesize that loss of IRBIT may actively participate in carcinogenesis and IRBIT may then be considered as a tumour suppressor.

Targeting dementia Whereas IRBIT expression may be decreased in cancer cells, it is interesting to note that IRBIT is highly expressed in the brain and in particular in the hippocampus. This high level of IRBIT expression in the brain may have an impact in Alzheimer’s disease.

Alzheimer’s disease is a neurodegenerative disease characterised by the accumulation in neurons of intracellular fibrillary tangles and by the deposition in the brain of extracellular neuritic plaques, called Amyloid plaques. These plaques are composed of a small peptide, known as Amyloid-? (A?), that is secreted by the neurons of Alzheimer’s disease patients. At the molecular level, A? is produced by the cleavage of Amyloid-? precursor protein (APP). Recent studies have shown that APP, and the enzyme which cleaves APP into A?, are localised in MAMs. Moreover, the analysis of cells from mouse models, and from actual Alzheimer’s disease patients, reveals that the amount of ER in contact with mitochondria is more abundant – and that the biological activity in these contact points is higher when compared to normal cells. A new hypothesis is in fact now emerging that proposes a modification of MAMs abundance and activity may be responsible or at least actively participate in the progression of Alzheimer’s disease¹¹.

Given the role we described for IRBIT in the promotion of ER-mitochondria contact, some might legitimately wonder whether IRBIT can be involved in Alzheimer’s disease. The idea may be strengthened by the fact that during Alzheimer’s disease, memory loss and cognitive impairment are consecutive to the death of neurons by apoptosis that induces the atrophy of several regions of the brain – notably the hippocampus where IRBIT is highly expressed. Then by promoting apoptosis and MAM formation, IRBIT appears as a possible contributor to Alzheimer’s disease progression.

New therapy? This new and unexpected role of IRBIT in apoptosis and MAMs formation opens new areas of research. Indeed, our results suggest a possible involvement of IRBIT in cancer and Alzheimer’s disease.

The deeper understanding of the mechanisms underlying these functions and notably the identification of the means by which IRBIT regulates ER-mitochondria contact represent an exciting challenge as this could offer a new therapeutic approach for cancer and neurodegenerative diseases.

Author: Dr Benjamin Bonneau is a research fellow at the Brain Science Institute in Wako-shi, Japan. He studies calcium signalling.

References:

1. Berridge, M. J. et al. Calcium signalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4, 517–29 (2003). 2. Mikoshiba, K. IP3 receptor/Ca2+ channel: from discovery to new signaling concepts. J. Neurochem. 102, 1426–46 (2007). 3. Bonneau, B. et al. Non-apoptotic roles of Bcl-2 family: The calcium connection. Biochim. Biophys. Acta - Mol. Cell Res. 1833, 1755–1765 (2013). 4. Giorgi, C. et al. Structural and functional link between the mitochondrial network and the endoplasmic reticulum. Int. J. Biochem. Cell Biol. 41, 1817–27 (2009). 5. Carson, D. A. & Ribeiro, J. M. Apoptosis and disease. Lancet (London, England) 341, 1251–4 (1993). 6. Ando, H. et al. IRBIT: A regulator of ion channels and ion transporters. Biochim. Biophys. Acta - Mol. Cell Res. 1843, 2195–2204 (2014). 7. Bonneau, B. et al. IRBIT controls apoptosis by interacting with the Bcl-2 homolog, Bcl2l10, and by promoting ER-mitochondria contact. Elife 5, 1–27 (2016). 8. Wittig, R. et al. Candidate genes for cross-resistance against DNA-damaging drugs. Cancer Res. 62, 6698–705 (2002). 9. Jeong, W. et al. Paradoxical expression of AHCYL1 affecting ovarian carcinogenesis between chickens and women. Exp. Biol. Med. 237, 758–767 (2012). 10. Arnaoutov, A. & Dasso, M. IRBIT is a novel regulator of ribonucleotide reductase in higher eukaryotes. Science (80-. ). 345, 1512–1515 (2014). 11. Area-Gomez, E. & Schon, E. A. Mitochondria-associated ER membranes and Alzheimer disease. Curr. Opin. Genet. Dev. 38, 90–96 (2016).

This work was supported by the Japan Society for the promotion of Science and by the RIKEN SPDR program.