Turning the spotlight on lipids in non-apoptotic cell death - ACS

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Turning the spotlight on lipids in non-apoptotic cell death Laura R. Parisi, Lauren M. Morrow, Michelle B. Visser, and G. Ekin Atilla-Gokcumen ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b01082 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on January 30, 2018

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Turning the spotlight on lipids in non-apoptotic cell death Laura R. Parisi1, Lauren M. Morrow1, Michelle B. Visser2, G. Ekin Atilla-Gokcumen1* 1

Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY 14260, USA 2

Department of Oral Biology, School of Dental Medicine, University at Buffalo, The State University of New York, Buffalo, New York 14260, USA

Abstract Although apoptosis has long dominated the spotlight, studies in the last two decades have expanded the repertoire of programmed cell death (PCD). Several forms of non-apoptotic regulated cell death have been identified, with important links to organismal homeostasis and different disease pathologies. Necroptosis, ferroptosis, pyroptosis and NETosis are the major forms of PCD that have attracted attention. Clear biochemical distinctions differentiate these forms of non-apoptotic PCD at the protein and membrane levels. For instance, pore formation at the plasma membrane is a hallmark of necroptosis and pyroptosis; however, different proteins facilitate pore formation in these processes. Here, we will highlight the role of lipids in different forms of non-apoptotic PCD. In particular, we discuss how lipids can trigger or facilitate the membrane-related changes that result in cell death. We also highlight the use of small molecules in elucidating the mechanisms of non-apoptotic PCD and the potential of lipid biosynthetic pathways to perturb these processes for therapeutic applications as a future avenue of research.


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Introduction Programmed cell death (PCD) is essential for maintaining cellular homeostasis and can be activated by various pathological and physiological states. Imbalance between cell proliferation and PCD has been linked to many diseases, including cancer, neurodegenerative disorders, autoimmune diseases, and viral infections1. Given the involvement of cell death in different diseases, understanding how these processes are triggered and facilitated by different biomolecules is of great therapeutic interest. The most widely recognized form of PCD is apoptosis. Apoptosis was initially characterized in 1972 by some of its distinct morphological features: cell shrinkage, nuclear and cytoplasmic condensation, and formation of apoptotic bodies2. For many years, apoptosis was considered the only form of PCD, while necrosis was thought of as ‘accidental’ death3. However, accumulating evidence in recent years has revealed the existence of many different forms of non-apoptotic PCD, including necroptosis, ferroptosis, pyroptosis, NETosis, autosis, entosis, and parthanatos. Among these, necroptosis, ferroptosis, pyroptosis are currently the best characterized. Distinct biochemical machineries regulate these different forms of PCD, each of which involves unique membrane-related changes. In the case of apoptosis, these changes include membrane blebbing and translocation of phosphatidylserine to the outer leaflet of the plasma membrane. In contrast, loss of plasma membrane integrity occurs during necroptosis, pyroptosis, and NETosis. Due to the loss of plasma membrane integrity, nonapoptotic PCD can cause inflammation at the organismal level3. Lipids are essential biomolecules that are well recognized for their structural roles in cellular membranes. However, they have also recently been recognized as important signaling molecules and key regulators of processes such as cell division4,5, senescence6,7, and cell death8-10. The mammalian lipidome consists of thousands of unique species, deriving its chemical diversity from different core structures, head groups, and acyl chains (which can vary in length and number and position of double bonds). These structural differences give rise to unique physiochemical properties for each lipid species, affecting their cellular localization, interactions within the cell, and the physical properties of the membrane in which they are present11,12. Despite recent advancements in tools and techniques to study lipids at the molecular level, the functions of individual lipids species in many cellular processes remain largely unknown. In the context of cell death, membrane-related morphological changes raise many questions. How does the cell utilize lipids during cell death? Are lipid species linked to specific signaling pathways? Are lipids involved in triggering cell death, or do they execute cell death by facilitating membrane-related changes? In this review, we will discuss the involvement of lipids in different forms of non-apoptotic PCD. We focus in particular on necroptosis, ferroptosis, and pyroptosis, which are of broad interest as they occur in multiple cell types and disease models. These forms of PCD involve specific membrane-related changes, and the roles of lipids in these processes have been the subject of previous reviews13,14. Here we provide an updated overview on the roles of lipids during necroptosis, ferroptosis, and pyroptosis, and also discuss potential roles of specific lipids during NETosis, which is less well characterized but may involve drastic remodeling of cellular membranes. We will also highlight the use of small molecules to examine the molecular mechanisms of non-apoptotic death programs, and how they have helped to elucidate the roles of lipids in these processes.


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NECROPTOSIS Discovery and molecular mechanisms. Necroptosis is a form of regulated cell death that results in hallmark features of necrosis including cellular swelling, loss of plasma membrane integrity, and the release of intracellular contents. Traditionally, necrotic cell death was considered to be unregulated and apoptosis was considered the sole form of programmed cell death. However, in the late 1980s, observations that death receptor activation could result in necrotic cell death led researchers to speculate that a different category of cell death might exist15. Several studies in the late 1990s showed that inhibition of caspases promoted necrotic cell death (reviewed in 16). In 2000 Holler et al. demonstrated that receptor interacting protein 1 (RIP1), which had been previously been linked to death receptor signaling, was necessary for caspase-independent necrotic cell death induced by death receptor ligands17. In 2005, Degterev et al. identified a small molecule inhibitor of RIP1-dependent, caspase-independent necrotic cell death called necrostatin-1 (Figure 1), and coined the term necroptosis to describe this pathway18. Later studies elucidated other key regulatory proteins involved in necroptosis including receptor interacting protein 3 (RIP3) (for a review of these discoveries, see16) and mixed lineage kinase domain-like protein (MLKL), which was identified via immunoprecipitation studies with a small molecule inhibitor, necrosulfonamide19 (Figure 1). A number of different agents induce necroptosis20; however, the molecular mechanisms have perhaps been most well defined for tumor necrosis factor (TNF)-induced necroptosis. Briefly, when death receptor TNFR1 is activated and pro-survival nuclear factor-κB activation is suppressed, a cytosolic complex containing RIP1, RIP3 and caspase-8 (among other proteins) can form (reviewed in21). Under these conditions, caspase-8 activity governs whether a cell will undergo apoptosis or necroptosis. If caspase-8 is active, it cleaves RIP122 and RIP323, preventing their activation, and apoptosis proceeds. If caspase-8 is inactive, however, a complex called the necrosome containing RIP1, RIP3, and MLKL forms19. Within this complex, RIP3 phosphorylates MLKL, which promotes MLKL oligomerization and translocation to the plasma membrane (Figure 2), a step that is essential for the execution of necroptosis24. Recent evidence suggests that MLKL forms pores or cation channels at the plasma membrane which disrupt osmotic balance, leading to the eventual rupture of the plasma membrane25. Emerging roles of PIPs and other lipids. Given the important membrane-related changes that occur during necroptosis, it comes as no surprise that lipids have been shown to play key roles in this process. MLKL has been shown to interact with a number of different phosphatidylinositol phosphates (PIPs)26,27 and also with cardiolipin27 (diphosphatidylglycerolipids that are primarily located in the inner mitochondrial membrane in mammalian cells). MLKL induces leakage of liposomes containing PIPs or cardiolipin, but not with liposomes containing phosphatidylinositol or phosphatidylserine27. Furthermore, small molecule inhibitors of PIKfyve and phosphatase and tensin homolog (PTEN) reduce cell death due to TNF-induced necroptosis26, affirming the functional role of these lipids during necroptosis and suggesting that perturbation of PIPs can modulate necroptotic activity. High-resolution structural characterization of MLKL oligomerization and membrane association suggested a multistep process28. The first step involves oligomerization facilitating a low-affinity binding to the plasma membrane. At the membrane, N-terminal helix bundle domain of MLKL undergoes structural arrangement which exposes binding sites on the domain that have high affinity to PIPs. Experiments in micelles


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investigating the interaction of N-terminal helix bundle domain with different PIPs suggested a binding preferences towards PI(4,5)P2 in the presence of PI and PI(4)P28. In addition to PIPs, other lipids are beginning to be recognized to have potential roles during necroptosis. Our laboratory recently conducted the first untargeted metabolomics study to investigate changes in the lipidome during necroptosis9. We found that very long chain fatty acids (VLCFAs) were among the most significant changes in the lipidome during necroptosis. Fatty acid synthase (FASN), and elongation of very long-chain fatty acid proteins 1 and 7 (ELOVL1 and ELOVL7) were upregulated during necroptosis, suggesting that fatty acid biosynthesis and elongation are responsible for VLCFA accumulation observed during necroptosis. Knockdown of FASN9 or ELOVL1/7 (unpublished data) via shRNA also prevented necroptotic cell death. We also demonstrated that a small molecule inhibitor of FASN, cerulenin, prevented VLCFA accumulation, cell death, and membrane permeabilization due to necroptosis9. Together, these findings firmly indicate that VLCFA play a functional role during necroptosis. Based on our observations, changes at the lipid level occur downstream to activation of the death pathway. As such, we think these lipids do not induce necroptosis but rather may be involved in the membrane-related changes that facilitate cell death by altering lipid and/or protein organization in the plasma membrane, either by themselves or potentially through incorporation into more complex lipids such as PIPs. Ceramides have also been linked to necroptosis, although the nature of their involvement is still an open question. Both we9 and others29,30 have found ceramides to accumulate during necroptosis. However, the mechanism resulting in the accumulation of ceramides or other up- or down-stream sphingolipids during necroptosis is yet to be determined. Some studies have suggested that cell death is mediated by increased ceramide levels as a result of sphingomyelinase activity29,31. Another recent study, on the other hand, has suggested sphingosine and sphingosine-1-phosphate (for a review on the roles of these signaling lipids, see32) as the mediators of cell death using a ceramidase knockout model33. It is possible that multiple pathways could be activated in different cellular contexts. Necroptosis has been linked to a number of different disease states. For example, it has been shown that many different bacteria induce cell death in macrophages via pore-forming toxins which induce necroptosis, contributing to bacterial pneumonia34. Upregulation of proteins involved in the necroptotic machinery such as RIP3 have been associated with of inflammatory diseases such as ulcerative colitis35, while suppression of RIP336 and MLKL37 have been associated with cancer. Treatment with RIP1 inhibitor necrostatin-1 or its more selective analogue Nec-1s (Figure 1) has ameliorated a number of different pathologies including allograft rejection38, ischemia/reperfusion injuries18 and neurodegenerative diseases39. However, we note the pharmacokinetic and pharmacodynamic limitations of necrostatin-140, and suggest that future studies using compounds with higher selectivity will be key to establishing the role of necroptosis in these pathologies. Given the importance of lipids during necroptosis, it will be crucial to evaluate how perturbing lipid biosynthesis modulates necroptosis in disease models. Our data, for instance, suggests that perturbing VLCFA biosynthesis may delay necroptotic cell death9. Currently, no selective inhibitors of individual ELOVLs exist, but their development could lead to better understanding of the roles of individual lipids during necroptosis and other processes, as well as provide potential therapeutic applications.


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FERROPTOSIS Discovery and molecular mechanisms. Ferroptosis is an iron-dependent form of cell death in which glutathione, oxidative stress and lipid peroxidation play crucial roles. As early as the 1970s, it was appreciated that dysregulation of intracellular iron can cause cell death. While studies in the subsequent years showed that iron chelators and a lipophilic antioxidant (vitamin E) could prevent cell death due to glutathione depletion (reviewed in41), it was not until very recently that ferroptosis was recognized as a unique form of cell death. The identification and characterization of ferroptosis was pioneered by the Stockwell laboratory. These investigations stemmed from key studies in the early to mid-2000s that aimed to screen for small molecules with genotype-selective toxicity toward oncogenic RAS42,43. Among the compounds they identified, two novel small molecules, erastin and RSL3 (Figure 1), induced non-apoptotic cell death. Studies to investigate the mechanism of action of these compounds showed that erastin and RSL3-induced cell death was not caspase dependent, nor did erastin-induced cell death result in typical morphology changes characteristic of apoptosis or necrosis43-45. Intriguingly, however, iron chelators and vitamin E prevented erastin and RSL3-induced cell death43, suggesting a crucial role for reactive oxygen species (ROS) in this process, which was named ferroptosis by Dixon et al. in 201245. Dixon et al. also showed that ferrostatin-1 (Figure 1), a small molecule identified to inhibit erastin-induced ferroptosis, prevented accumulation of cytosolic ROS and lipid peroxidation.45 Initial studies to determine the target of erastin highlighted mitochondrial voltage-dependent anion channel proteins (VDACs)44, which were suggested as potential mediators of ROS during ferroptosis. Indeed, shRNA knockdown of VDAC3 can prevent loss of cell viability due to erastin-induced ferroptosis, albeit only to a certain extent and in specific cell lines44,45. However, another mechanism of action was soon identified. Small molecule-based modulatory profiling led to the discovery that erastin is an inhibitor of system xc- 45,46, a cell surface cystine-glutamate antiporter. Metabolomic profiling revealed that erastin treatment results in a depletion of glutathione47, presumably due to limited availability of cysteine as a result of system xcinhibition since the rate limiting step of glutathione biosynthesis is the condensation of cysteine with glutamate48 (Figure 2). Erastin treatment was then shown to decrease the activity of glutathione-dependent peroxidases (GPXs)47, which can reduce organic peroxides such as lipid peroxides to hydroxides49. The same study showed that RSL3 directly binds and inhibits the peroxidase activity of GPX4, and knockdown of GPX4 strongly sensitized cells to RSL3induced ferroptosis while overexpression of GPX4 conferred resistance to cell death, finally providing a unified underlying mechanism to begin to understand how these molecules induce ferroptosis (Figure 2). Fundamental role of lipid peroxidation. It seemed likely from quite early on that lipids may be involved during ferroptosis, and evidence continues to accumulate that lipid peroxidation is integral to this process. Proteins that mediate ferroptotic cell death are directly involved in lipidrelated pathways. Inactivation of GPX4 is central to erastin and RSL3-induced ferroptosis. GPX4 utilizes glutathione to reduce lipid peroxides, a function which is important to prevent propagation of oxidative damage to other lipids and biomolecules49. Gpx4 knockout in mice is embryonic lethal50 and inducible knockout of Gpx4 causes ferroptosis accompanied by increased levels of lipid hydroperoxides51. In addition to GPX4, aldo-keto reductase AKR1C family enzymes, which may also play a role in the detoxification of products of lipid peroxidation52,


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may be involved in ferroptosis. Erastin/RSL3 resistant clonal cell lines selected by maintained high doses of erastin showed >500-fold increases in expression of two AKR1C family genes46. Lipoxygenases53, on the other hand, promote ferroptosis. Knockdown of arachidonic lipoxygenases (ALOXs) has been shown to reduce sensitivity to erastin-induced ferroptosis54. However, the context in which lipoxygenases participate in ferroptosis is not entirely clear and may be dependent on glutathione levels54 or other potential mediators such as phosphatidylethanolamine binding protein 1, which has been shown to interact with 15lipoxygenases under certain conditions and may promote oxidation of 55 phosphatidylethanolamines . In addition to proteins involved in lipid peroxidation, proteins involved in lipid biosynthesis play key roles in ferroptosis. Genetic screens revealed that loss of function of lipid biosynthesisrelated genes, in particular acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3), confers a survival advantage during ferroptosis56. ACSL4 preferentially activates PUFAs57 for synthesis of complex lipids (ex. phospholipids and glycerolipids), which are more susceptible to lipid peroxidation than monounsaturated or saturated fatty acids. Acsl4 knockout cells are highly resistant to ferroptosis 58,59 , and Lpcat3 knockdown also confers resistance to ferroptosis, although to a lesser extent58. These findings suggest that PUFA-containing phospholipids play a key role in ferroptotic cell death. Indeed, several lipidomic studies have shown that ferroptosis is accompanied by changes in phospholipid composition. Inducible knockout of Gpx4 caused ferroptotic cell death, and increased levels of hydroperoxy-fatty acids, -phosphatidylcholines, -phosphatidylethanolamines, and –cardiolipins as well as increases in lysophosphatidylcholines and 51 lysophosphatidylethanolamines in mouse kidneys . A lipidomic analysis of cells treated with an erastin analogue found depletion of PUFA-containing phosphatidylcholines and an increase in lysophosphatidylcholine among the most significant changes in the lipidome54. In another recent study, oxidized phospholipid species were detected between wild type, Gpx4 knockout, and Acsl4 knockout mouse embryonic fibroblasts during RSL3-induced ferroptosis. Among these species, the authors proposed phosphatidylethanolamines containing arachidonic acid (C20:4) or adrenic acid (C22:4) as key drivers of ferroptosis since their levels correlated with degree of cell death58. The precise chemical structures of different lipids influence their susceptibility to oxidation, and it is clear that structure has a functional impact on the roles of lipids during ferroptosis. A recent study that investigated the role of lipoxygenases in ferroptosis showed the modulatory effect of a number of different lipids on RSL3 treatment. It was found that oleic acid (a monounsaturated fatty acid) strongly suppressed ferroptotic cell death, while arachidonic acid and linoleic acid (polyunsaturated fatty acids) sensitized cells to ferroptotic cell death54. The authors also showed that labeling linoleic acid with deuterium in the bis-allylic position, which slows down the rate of peroxide formation, prevented generation of lipid peroxides and ferroptotic cell death54. Another study which suggested key involvement of ACSL4 in ferroptosis showed that addition of ω-6 fatty acids generally sensitized Acsl4 knockout cells to ferroptosis, while ω-3 fatty acids had little to no effect59, with the exception of docosahexaenoic acid. It is clear that lipid peroxidation plays a central role in ferroptosis; however, several major questions remain to be answered. The precise mechanism(s) by which lipid peroxidation result in ferroptotic cell death still need to be elucidated. Although lipid peroxidation can significantly


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alter membrane fluidity and permeability, it is possible that products of lipid peroxide degradation such as malondialdehyde and 4-hydroxynonenal react with other biomolecules including protein and DNA60, exerting toxic effects. Similarly, despite iron-dependence being a defining characteristic, the role of iron during ferroptosis is also still not thoroughly understood. Ferroptosis can be inhibited by a number of iron chelators and has been shown to be modulated by the availability of transferrin-iron complexes61. However, it is unclear whether ferroptosis proceeds via production of hydroxyl radicals through Fe2+ or iron-dependent lipid processing enzymes62. Ferrostatin-1 (Figure 1), which was shown to prevent cell death in Huntington’s and other disease models, has been shown to prevent depletion of PUFAs due to erastin treatment, without affecting glutathione levels63. Besides ferrostatin-1, a number of classes of antioxidants are able to inhibit ferroptosis with similar or better potency which correlates with their ability to prevent oxidation of liposomes64. It seems clear that ferrostatin-1 prevents ferroptosis by preventing peroxidation of PUFAs and/or other PUFA-containing lipids45,63 due to its function as a lipophilic antioxidant65. Thus far, ferroptosis has been linked to pathological processes including ischaemia/reperfusion injuries and certain neurodegenerative diseases, and a few studies have suggested a potential role in tumor suppression66,67. While apoptosis has well established roles in development and tumor suppression, and other forms of regulated cell death (i.e. necroptosis, pyroptosis, and NETosis) have links to defense against pathogens, a beneficial role of ferroptosis at the organismal level has yet to be elucidated. PYROPTOSIS Discovery and molecular mechanisms. Pyroptosis is an inflammatory, caspase-1-dependent programmed cell death which primarily occurs in macrophages and neutrophils68,69. Due to its caspase-dependence−at the time, a hallmark of apoptosis−initial observations of pyroptotic death in infected macrophages were misidentified as apoptosis69,70. Although some phenotypic features of pyroptosis overlap with those of other cell deaths, clear biochemical distinctions established pyroptosis as an independent death pathway. Studies with Salmonella typhimurium revealed the inflammatory nature and caspase-1 dependence of pyroptosis71, distinguishing it from noninflammatory apoptotic pathways. DNA damage occurs during both apoptosis and pyroptosis, but pyroptotic cells maintain nuclear integrity and display a different DNA fragmentation pattern from apoptotic cells71. Similar to necroptotic cells, pyroptotic cells exhibit plasma membrane permeabilization and release proinflammatory molecules, in contrast to the non-inflammatory removal of apoptotic bodies during apoptosis3,69,71. These distinct features lead Cookson et al. to aptly name the pathway pyroptosis in 2001, derived from the Greek root pyro-, meaning “fire” in reference to its inflammatory nature72. Following initial characterizations, other recently described phenotypic changes include membrane blebbing prior to plasma membrane rupture and the release of fragments of the cell body, named pyroptotic bodies73. Due to its inflammatory nature, pyroptosis has been observed in many disease and injury states69. At the cellular level, pyroptosis is initiated upon activation of pattern recognition receptors (PRRs) (reviewed in68,69, see Figure 2). Cells use a variety of these receptors, including membrane-bound C-type lectin and Toll-like receptors, as well as intracellular receptors such as NOD-like receptors (NLRs) and DNA-sensing absent in melanoma 2 (AIM2). When these receptors detect signals generated either by the host cell in response to stress or tissue injury or by invading pathogens, they initiate signaling cascades (reviewed in74). Some PRRs including AIM2, the protein pyrin, and the Nod-like receptors Nlrp3, Nlrp1b, and Nlrc4, assemble


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inflammasomes (Figure 2)68,74. These multiprotein complexes recruit and activate caspase-1 through adaptor proteins such as ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain)74. Hung and coworkers elucidated the mechanism of inflammasome assembly during pyroptosis by using the small molecule 7DG (Figure 1), which suppressed cell death. They identified protein kinase R as the target of 7DG, placing protein kinase R as a central molecule in inflammasome assembly and caspase-1 activation during pyroptosis75. While dozens of protein targets exist for caspases during apoptosis, evidence indicates that only a few caspase targets are necessary for the progression of pyroptosis. One key protein target in the pyroptotic pathway is Gasdermin D (GSDMD)76. Activated caspase-1 facilitates pyroptosis through cleavage of GSDMD to generate an N-terminal cleavage product (GSDMD-NT), which oligomerizes and translocates to the plasma membrane, where it forms pores68,77,78. In addition, activated caspase-1 matures pro-IL-1β to its cleaved active form. IL-1β is subsequently released from the cell along with other proinflammatory mediators through the GSDMD-NT-mediated plasma membrane pores79. The transmembrane flux of molecules, including ions and cytokines, through the plasma membrane pores ultimately results in lysis of the cell73. Although plasma membrane permeabilization is characteristic of both pyroptosis and necroptosis, there are differences in how the permeabilization facilitates cell death. These differences can be linked to properties of the pores generated by GSDMD-NT and MLKL. Studies have suggested that MLKL forms selective cation channels which generate osmotic imbalance, resulting in the necroptotic hallmarks of cellular swelling and plasma membrane rupture25. In contrast, pores formed by GSDMD-NT lack ion selectivity73. As a result, change in osmotic pressure should have less effect during pyroptosis compared to necroptosis73. Roles of lipids in pyroptosis. Recent studies have suggested that lipids play roles in pyroptosis, both as triggers and facilitators of the membrane-related phenotypes involved in this process. A specific oxysterol80 and fatty acid81 have been reported to activate pyroptotic cell death. 25hydroxycholesterol activates P2X7, an ion channel, and caspase-1, resulting in the release of inflammatory cytokines via pyroptosis80,82. Pillon et al. have shown that palmitate, a saturated fatty acid, increases the activity of inflammatory caspases and induces membrane permeabilization and the release of inflammatory cytokines, indicating pyroptotic activity81. In contrast, unsaturated fatty acids palmitoleate and oleate did not elicit inflammatory responses or toxicity81. In addition to their roles in activating the pyroptosis pathway, lipids are also likely involved in facilitating the membrane related changes occurring during pyroptosis. Since interaction with membrane lipids might affect membrane localization and pore formation, Liu et al.77 and Ding et al.78 investigated potential interactions between GSDMD-NT and certain membrane lipids. Consistent with previous findings that GSDMD-NT facilitates membrane permeabilization during pyroptosis76, full length GSDMD, mutant GSDMD-NT77, and GSDMD-CT do not interact with membrane lipids, while GSDMD-NT shows lipid binding77,78. Studies in liposomes showed that GSDMD-NT could bind to PIPs, cardiolipin, phosphatidic acid (PA) and phosphatidylserine (PS) species77,78, but not to phosphatidylethanolamine or phosphatidylcholine77. Interestingly, each of the lipids shown to interact with GSDMD-NT is net anionic under physiological conditions, which likely contributes to the binding interactions.


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Additionally, while PIPs, PA, and PS are components of the inner leaflet of the plasma membrane in mammalian cells, cardiolipins are major components of the inner mitochondrial membrane in mammalian cells12 and are also present in bacterial membranes83. As such, these findings indicate that GSDMD-NT binds to the inner mammalian plasma membrane, and potentially to mitochondrial or bacterial plasma membranes77. However, these interactions remain to be demonstrated in vivo. Intriguing questions regarding the molecular interactions between GSDMD-NT oligomers and membrane lipids also remain to be elucidated, including how the electrostatic properties of the lipid head group, surface properties of the protein and overall lipid composition of membranes mediate these interactions. Additionally, further studies are needed to identify any lipid metabolic enzymes that are required for the progression of pyroptosis, similar to the chemical and genetic screens that helped to elucidate the crucial involvement of lipid peroxidation during ferroptosis. NETosis Discovery and characterization. Neutrophils are a type of immune cells which are able to bind, engulf, and kill invading pathogens84. As terminally differentiated cells, neutrophils have a short life span, but have been shown to extend their antimicrobial activity through the release of extracellular traps85. Neutrophil extracellular traps (NETs) are potent antimicrobial structures released by neutrophils to the extracellular environment which can bind and kill various bacteria, viruses, and fungi86. NETs were first described in 2004, when researchers observed that neutrophils released networks of thread-like structures in response to certain stimuli84. These structures contained histone-rich chromatin backbones decorated with antimicrobial granule proteins, which were able to bind and kill pathogens including gram-negative and -positive bacteria84. Following initial characterizations, NET formation and release was proposed to be part of a novel cell death program (later named NETosis87) due to distinct characteristics including a lack of DNA fragmentation and disintegration of the nuclear membrane85. Emerging evidence indicates that NETosis may progress through distinct signaling pathways that are stimulus-dependent88. Classically, NETosis has been described as an NADPH oxidase (Nox)dependent death program which results in NET formation/release facilitated by compromised cellular membranes. To date, the mechanistic details of pathways involved in NETosis have not been well characterized89, but seem to involve protein kinase C and Raf-MEK-ERK pathways90,91. NETosis involves intriguing morphological changes which have captured by live cell imaging and transmission electron microscopy studies85. At the onset of NETosis, activated neutrophils initially flatten and form intracellular vacuoles. Subsequently, nuclei expand and lose their characteristic lobular shape, and chromatin decondensation occurs. Granular materials become distributed throughout the cytoplasm and the nuclear membrane disintegrates, allowing decondensed chromatin to interact with cytoplasmic and granular materials, thus allowing NETs to form. Eventually the plasma membrane ruptures, allowing the NETs to be released from the cell body85. In addition to Nox-dependent NETosis, other pathways involving NET formation and release have also been described. Calcium-selective bacterial ionophores, which bind and transport cations across cell membranes, have been shown to induce release of NET-like structures in a Nox-independent manner88,92. Furthermore, recent reports indicate that certain stimuli can induce


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“vital NETosis”, which involves release of either nuclear or mitochondrial DNA in the absence of cell lysis (reviewed in93). Although NETosis has been considered a beneficial antimicrobial mechanism, dysregulated NETosis may also lead to the development and exacerbation of inflammatory diseases86. It is therefore important to fill the gaps in knowledge concerning how different biomolecules, including lipids, facilitate the morphological changes that lead to the formation of extracellular traps. Roles of lipids in NETosis. Membranes play important roles during NETosis at multiple stages. First, the nuclear envelope disintegrates to allow chromatin to interact with granular proteins and cytoplasmic materials to form NETs. Second, the plasma membrane ruptures releasing the NETs, along with other intracellular material, to the extracellular matrix. Given that lipids can modulate the biophysical properties of membranes12, it is reasonable to expect that specific lipids and changes in lipid composition may contribute to the membrane-related changes during NETosis. Recent studies have shown that NET formation is sensitive to the cellular levels of an important membrane lipid, cholesterol. Treatment with statins94 and methyl-β-cyclodextrin95 to reduce cholesterol levels in neutrophils resulted in increased NET formation and release. Cholesterol, together with other lipids, are thought to be important signaling platforms in various cellular events, including host-pathogen interactions96. Based on this it is plausible that cholesterol, in combination with other lipids, could influence the physical properties of membranes (i.e., overall membrane fluidity) or promote specific signaling events via membrane domains in activated neutrophils and facilitate their rupture. Another lipid which may potentially regulate NETosis is Hepoxilin A3. Hepoxilin A3 is a proinflammatory, oxidized eicosanoid secreted by epithelial cells in response to bacterial infection that recruit neutrophils to the site of infection. Neutrophils exposed to Hepoxilin A3 or a synthetic analog have been shown to release DNA to the extracellular environment in a doseand time-dependent manner97. However, we are only just beginning to understand the roles of lipids during NETosis. The signaling processes and downstream events driving NET formation and release are only partially characterized. As such, there is certainly a need for further mechanistic understanding of NETosis, including the roles of lipids in this process. In this regard, we expect that future studies to investigate changes in the lipidome or localization of specific lipids during different stages of NETosis may provide critical insights. CONCLUSIONS Over the past decade it has become clear that there are many pathways by which our cells can die, each of which involves unique membrane-related phenotypes where lipids play important roles. In some cases, specific lipids can cause the activation of these pathways. In other cases, however, lipids seem to function downstream of the activation of protein machinery, where distinct changes in lipid composition and/or protein-lipid interactions have critical functions in the execution of cell death. The time is ripe to ask ourselves: can we harness the functions of lipids to perturb cellular processes including cell death? Studies beginning to explore lipid-centered therapeutic strategies have indeed demonstrated the potential to modulate cell death in vivo. For example, nanoliposomes containing C6-ceramide


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(which were shown to induce MLKL-dependent necrotic cell death in vitro) were shown to suppress metastasis in a mouse xenograft model98. However, a great deal of work remains in order to understand the precise contributions of different forms of cell death in vivo. For instance, although apoptosis is often considered the primary form of cell death induced by most chemotherapeutic agents, is it the sole mechanism of tumor cell death? Could other forms of cell death provide an alternative strategy for treating apoptosis-resistant tumors? Certain chemotherapeutic agents have been shown to induce necroptosis20. Furthermore, it has recently been shown that secondary necrosis (late-stage necrosis that occurs in apoptotic cells), may in some cases be a regulated process which is executed by caspase-3 dependent cleavage to release the N-terminal fragment of gasdermin superfamily protein DFNA5/GSDME99. The underlying similarity of highly regulated pore formation events between necroptosis, pyroptosis, and potentially secondary necrosis is intriguing. Further investigations into the differences between these events are highly warranted, particularly in regard to how interactions with lipids enable these proteins to form pores in cellular membranes. Small molecule-based approaches have been fundamental in understanding the mechanisms of different cellular processes, and have been particularly instrumental in elucidating different forms of non-apoptotic PCD. For instance, chemical screens identifying erastin and RSL3 led to the identification of ferroptosis as unique form of cell death. Similarly, the identification of RIP1 inhibitor necrostatin-1 helped to solidify necroptosis as a distinct, regulated form of nonapoptotic cell death18. In the case of pyroptosis, small molecule 7DG, which was also identified via a chemical screen, helped to elucidate some aspects of inflammasome assembly and caspase1 activation75. Given the important roles of lipids in these different forms of cell death, small molecules targeting lipid biosynthesis have begun to emerge as important tools for understanding downstream events in these pathways, and as potential therapeutic agents. Small molecules targeting fatty acid9 and PIP26 biosynthesis have been shown to modulate necroptosis, while small molecule inhibitors of ACSL4 have been shown to protect against ferroptosis59. Different forms of PCD are implicated in diverse pathologies ranging from neurodegenerative diseases, pathogen infection, inflammatory conditions and autoimmune diseases. Efforts to understand the contributions of different forms of PCD in disease are growing in parallel with the awareness that lipids play important roles in these processes. Enzymes related to lipid biosynthesis and processing are evolved to process and could therefore be targeted by small molecules. However, because our understanding of the roles of individual lipids is limited, these enzymes have not been prioritized for drug discovery yet. As our understating of the involvement and regulation of different lipids in PCDs advances, we expect that the development of lipid-centered therapeutic strategies will emerge as an exciting avenue of research.


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Keywords Necroptosis: A highly regulated form of necrotic cell death characterized by cellular swelling, plasma membrane rupture, and release of intracellular contents. PIPs: Phosphatidylinositol phosphates (PIPs) are a class of phospholipids that can be phosphorylated at multiple positions at the inositol head group. These lipids recruit proteins to the plasma membrane during necroptosis. PTEN: Phosphatase and tensin homolog dephosphorylates the 3rd position of PI(3,4,5)P3. PIKfyve: Phosphoinositide kinase preferentially phosphorylates the 5th position of PIs and PI(3)Ps. Ferroptosis: An iron-dependent form of programmed cell death that is characterized by the accumulation of lipid peroxides. Key protein regulators are involved in lipid biosynthesis and peroxidation. Pyroptosis: An inflammatory, caspase-1-dependent form of cell death characterized by DNA fragmentation, plasma membrane permeabilization and release of proinflammatory cytokines. Inflammasome: Multiprotein complexes that mediate the activation of proinflammatory caspases in response to pathogen- and host-derived ‘danger signals’. Inflammasomes activate caspase-1 to induce pyroptosis. NETosis: A form of cell death in which neutrophils form and release antimicrobial extracellular traps. NETosis is characterized by disintegration of nuclear and granule membranes, chromatin condensation, and release of neutrophil extracellular traps into the extracellular environment. Ceramidase: Ceramidase catalyzes the hydrolysis of ceramides into fatty acid and sphingosine species. Sphingomyelinase: Sphingomyelinase catalyzes the hydrolysis of sphingomyelins into ceramides.


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Figure 1. Small molecules used to elucidate the molecular mechanisms of non-apoptotic programmed cell deaths. A. Necrostatin-1, Nec-1s, and necrosulfonamide all inhibit necroptosis through the inhibition of key protein mediators. Necrostatin-1 and its more potent analog, Nec-1s, inhibit RIP1, while necrosulfonamide inhibits MLKL oligomerization. B. Erastin and RSL3 have been instrumental in identifying key mediators of the ferroptosis pathway including GPXs. Ferrostatin-1, an inhibitor of ferroptosis, highlighted the importance of lipid peroxidation during this process. C. The small molecule 7DG was discovered to inhibit pyroptosis through binding to PKR, which helped elucidate the role of PKR in inflammasome assembly and caspase-1 activation.


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Figure 2. Mechanisms of non-apoptotic programmed cell deaths. Necroptosis is a form of regulated necrosis characterized by cellular swelling, plasma membrane permeabilization, and release of intracellular contents. It can be initiated upon death receptor activation in the absence of caspase-8 activity, which leads to the activation of RIP1 and RIP3. Active RIP3 phosphorylates MLKL. MLKL oligomers translocate to the plasma membrane, where they interact with membrane lipids including PIPs. These interactions result in the permeabilization of the plasma membrane. Ferroptosis is an iron-dependent form of cell death in which oxidative stress and lipid peroxidation have crucial roles. It can be induced when GPX4 activity is impaired. This results in the oxidation of PUFAs or PUFA-containing phospholipids (upon activation and processing by ACSL4 and LPCAT3, respectively) and the formation of lipid peroxides. Pyroptosis is an inflammatory, caspase-1 dependent cell death that is characterized by loss of plasma membrane integrity and release of proinflammatory molecules. The process is initiated when certain pattern recognition receptors are activated and assemble inflammasomes, which often contain the adaptor protein ASC. The inflammasomes activate caspase-1, which proteolytically matures proinflammatory cytokines and cleaves GSDMD to release its Nterminus (GSDMD-NT). GSDMD-NT oligomerizes and translocates to the plasma membrane, where it interacts with membrane lipids and forms pores. NETosis is a form of programmed cell death in which activated neutrophils release thread-like chromatin structures covered with antimicrobial proteins, which bind and kill pathogens. At the onset of NETosis, activated neutrophils flatten, then chromatin decondensation occurs and nuclei expand and lose their lobular shape. Nuclear and granular membranes disintegrate, and decondensed chromatin mixes with granular and cytoplasmic proteins, forming NETs. Finally, the plasma membrane ruptures and NETs are released.


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