Necroptosis: how cells die after intracerebral hemorrhage

By Sarah Choi

There are a number of ways in which cells can die, morbid as that may seem. This includes the more commonly known apoptosis, a form of programmed cell death. Slightly more unknown is necrosis, which is, in many ways, thought to be the opposite of apoptosis. For example, unlike apoptosis, necrosis is caspase-independent, not genetically programmed, and leads to cell membrane rupture. Importantly, necrosis is a form of unregulated cell death. However, it has recently been found that there are forms of necrotic cell death that are regulated. These include necroptosis, ferroptosis, and mitochondrial permeability transition pore (MPTP)-mediated necrosis. It may be striking that necrosis and necroptosis seem to be the same word at first glance, and indeed, these two processes share multiple characteristics, including organelle swelling and plasma membrane rupture. In fact, necroptosis is an amalgam of “necrosis” and “apoptosis”, having features of both processes. As many diseases and conditions involve or result in cell death, understanding of these pathways is critical to developing treatments.

Necroptosis is a form of regulated cell death. It signals via similar pathways and proteins as necrosis, which are activated by either intracellular or extracellular triggers. For example, ischemia, the blockage of blood supply leading to insufficient oxygen and nutrient delivery, can lead to intrinsic necroptosis. Certain metabolic enzymes can also encourage ROS production, which promotes necroptosis. Extrinsic stimuli can act via a number of transmembrane receptors, including tumor necrosis factor (TNF) superfamily receptors. These triggers activate the receptor interacting protein kinase 3 (RIPK3) directly or via RIPK1. Activated RIPK3 then phosphorylates mixed lineage kinase domain-like protein (MLKL), which translocates to the plasma membrane. MLKL induces necroptosis by either recruiting sodium or calcium ion channels, or promoting pore formation (Dhuriya and Sharma, 2018; Molnár et al., 2019).  

The regulation of necroptosis is complex and as yet incompletely understood. Necroptosis is regulated by various proteins and enzymes and can be controlled by both caspase-dependent and caspase-independent mechanisms. Active caspases cleave RIPK1 and RIPK3, therefore apoptosis usually downregulates necroptosis. However, once RIPK3 dimerizes, necroptosis can be induced (Molnár et al., 2019). As there may be further signaling pathways involved – for example, there are other proteins that can functionally replace RIPK1 in the RIPK1/RIPK3 signaling pathway – there may be other forms of regulation that are unknown.

Recently, the role of necroptosis in intracerebral hemorrhage (ICH) was elucidated (Wei et al., 2021). ICH is a type of stroke with high mortality rates and severe consequences. ICH describes the rupture of blood vessels within the brain. The leakage of blood into tissues can lead to blood clots and inflammation. This leads to ischemia downstream of those vessels, and consequently, cell death. Secondary brain injury, marked by cell death, is the main cause of neurological impairment, and in ICH, this cell death was found to be necroptotic as opposed to previous suggestions of apoptosis and necrosis. Interestingly, this was related to microglial cells.

Microglia are neuroprotective immune cells in the central nervous system. They are activated by thrombin, an enzyme used to clot the blood, which also induces apoptosis in cultured neurons and astrocytes (Keep, Hua and Xi, 2012). As with thrombin, microglia have both beneficial and adverse effects. One of the functions of microglia cells is regulation of neuron excitability by the secretion of exosomes containing microRNAs (miRNAs). Exosomes are vesicles released from cells via fusion of a multivesicular body with the cell membrane. They can contain proteins, lipids, mRNAs, and miRNAs (Edgar, 2016). Exosomes are therefore thought to be a means of intercellular communication, allowing cells to influence their environment and mediate cell death, which, in the case of ICH, propagates necroptosis.

Through the use of rat models, Wei et al. revealed the role of microglia and miR-383-3p in ICH-induced necroptosis. Propidium iodide (PI) staining of dead or damaged cells, western blot detecting RIPK1, RIPK3, and MLKL expression, and flow cytometry was used to visualize and locate the site of necroptosis. RT-qPCR, co-culturing and rescue experiments, as well as an in vivo verification in ICH rats then helped reveal the mechanisms leading to cell death after ICH. They showed that miR-383-3p expression was significantly increased after ICH. Luciferase assay also demonstrated that the target gene of miR-383-3p was activating transcription factor 4 (ATF4). ATF4, with roles in cell survival and cell death, is often negatively correlated with RIP1, RIP3, and MLKL expression. miR-383-3p was found to bind the 3’-UTR of ATF4 to inhibit its expression. Therefore, ICH increases the necroptosis of neurons via activating microglia, that then secrete exosomes containing the miRNA miR-383-3p. miR-383-3p enters neurons, downregulates ATF4, and thereby promotes neural cell death by necroptosis via the RIPK1/RIPK3 pathway (Wei et al., 2021). This suggests targeting miR-383-3p may alleviate ICH-associated brain injury, as necroptosis of neurons will be reduced.

Apart from ICH, the pathogenesis of many neurodegenerative diseases, cardiovascular diseases, gastrointestinal diseases, and skin diseases involve necroptosis. This is due to the correlation between necroptosis and the loss of cells that is a major cause of the symptoms related to these diseases. Therefore, elucidating the mechanisms involved in necroptosis in these tissues and the regulatory mechanisms used would contribute to the development of novel treatments. Furthermore, necroptosis in cancer, with both beneficial and adverse effects, has been used in anti-cancer treatments. RIPK3 has also been suggested to be regulated by oncogenes (Molnár et al., 2019). Exosomes are involved in necroptosis in a variety of diseases, as well as other forms of cell death. Additionally, after ischemic stroke, microglia induce endothelial necroptosis as well (Wei et al., 2021), pointing to its role in the death of cells other than neurons. The variety of proteins, molecules, and mechanisms involved in necroptosis, and their linkage to other forms of cell death, results in a high level of diversity and complexity that is yet to be elucidated. Further characterization of these mechanisms can lead to novel treatments that reduce injury and alleviate symptoms.

References:

Dhuriya, Y. K. and Sharma, D. (2018) ‘Necroptosis: A regulated inflammatory mode of cell death’, Journal of Neuroinflammation. BioMed Central Ltd., pp. 1–9. doi: 10.1186/s12974-018-1235-0.

Edgar, J. R. (2016) ‘Q & A: What are exosomes, exactly?’, BMC Biology. BioMed Central Ltd., 14(1), p. 46. doi: 10.1186/s12915-016-0268-z.

Keep, R. F., Hua, Y. and Xi, G. (2012) ‘Intracerebral haemorrhage: Mechanisms of injury and therapeutic targets’, The Lancet Neurology. NIH Public Access, pp. 720–731. doi: 10.1016/S1474-4422(12)70104-7.

Molnár, T. et al. (2019) ‘Current translational potential and underlying molecular mechanisms of necroptosis’, Cell Death and Disease. Nature Publishing Group, pp. 1–21. doi: 10.1038/s41419-019-2094-z.

Wei, M. et al. (2021) ‘Activated Microglia Exosomes Mediated miR-383-3p Promotes Neuronal Necroptosis Through Inhibiting ATF4 Expression in Intracerebral Hemorrhage’, Neurochemical Research. Springer, 1, p. 3. doi: 10.1007/s11064-021-03268-3.

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