Distinguishing Non-Apoptotic Cell Death by Antibody-Based Methods

Introduction

It can be difficult to differentiate between different forms of non-apoptotic cell death, mainly because many of these have comparable morphological features. On the other hand, distinct regulatory pathways are involved with each form of non-apoptotic cell death providing distinct protein markers, which can then be used to detect them. This article shows how to distinguish the ferroptosis, necroptosis and pyroptosis processes.

Regardless of the mode of cell death being studied, it is best to employ a combination of various techniques. In order to rule out apoptosis, the analysis of non-apoptotic cell death should employ both specific positive indicators of the preferred cell death mode, together with other cell viability techniques and assays1.

Detecting necroptosis

When a ligand binds to death receptors, such as tumor necrosis factor receptor 1 (TNFR1)2, necroptosis is activated. While a number of proteins play a role in the necroptotic pathway, necroptosis can be reliably detected through specific inhibition of the necroptotic pathway and by measuring the status of MLKL phosphorylation.

 

Key proteins in the necroptotic pathway

Protein

Role in necroptosis

RIPK1

Protein kinase that recruits RIPK3 to the necrosome, leading to mutual phosphorylation of RIPK3 and RIPK1.

RIPK3

Protein kinase that phosphorylates MLKL3. It is activated by phosphorylation by RIPK1 and ensuing oligomerization.

MLKL

Kinase domain-like protein. After MLKL is phosphorylated by RIPK3, it translocates to the cell membrane to mediate cell death11.

Caspase-8

Inhibits necroptosis5.

 

Phosphorylation of MLKL

RIPK3-mediated phosphorylation activates MLKL, and its activation state can be established by measuring the Thr357 and Ser358 phosphorylation status. Antibody-based methods, such as flow cytometry, IHC, and western blot, are used for detecting phospho-MLKL.

Inhibition of necroptosis

Components of the necroptotic pathway can be targeted either with transgenic models or by chemical inhibition to determine if necroptosis is responsible for cell death.

 

Compound

Target

Necrostatin-1 (Nec1)

RIPK1

7-Cl-O-Nec-1 (Nec1s)

RIPK1

GSK'872

RIPK3

Necrosulfonamide

MLKL

 

Considerations when using inhibitors:

  • Nec1s is more specific, while Nec 1 possesses some off-target activity6.
  • RIPK1 can play a role in apoptosis7, but under certain conditions RIPK1 inhibitors can also block apoptosis.
  • The presence of necroptosis can be best confirmed with transgenic models.

 

Detecting pyroptosis

Pyroptosis, an inflammatory caspase-dependent form of programmed necrosis, takes place in response to infections caused by microbes. With regards to morphology, pyroptotic cells exhibit cell swelling and instant plasma membrane lysis. Pyroptosis can be examined by observing the gasdermin D cleavage and activation of caspase, or by ablating or inhibiting major components of the pyroptotic pathway.

 

Key components of the pyroptotic pathway

Protein

Function

Role in pyroptosis

Caspase 1

Inflammatory caspase, triggered by inflammatory agents and sensor proteins

Cleaves gasdermin D

Caspase 4 and 5 (human) or Caspase 11 (mouse)

Inflammatory caspases, triggered by bacterial polysaccharides

Cleaves gasdermin D

Gasdermin D

Cleaved by caspases8–10

Performs pyroptosis

 

Caspase activity

During pyroptosis, caspases that are active are cleaved from their inactive pro-caspase forms. Using a specific caspase antibody, cleavage of caspase can be identified by western blot.

Even though active caspases are cleaved, simply observing the caspase cleavage does not confirm the activation of caspase and therefore other techniques should also be employed to achieve pyroptosis confirmation.. Caspase activation assays can also be used for direct detection of caspase activation.

 

Gasdermin D

During pyroptosis, gasdermin D (53 kDa) is cleaved which results in a 30 kDa N-terminal fragment; western blot detects this fragment. We suggest using Abcam’s anti-gasdermin D rabbit polyclonal (ab155233) which is capable of detecting the N-terminal area of gasdermin D.

Inhibition of pyroptosis

In order to distinguish pyroptotic cell death from other forms of apoptosis and necroptosis, it is important to show dependence on caspase 1, 11, 4 or 5. Either transgenic models or chemical inhibition can be used to find out if cell death still occurs following ablation of these caspases. Ablation of caspase 1 activity can be done through chemical inhibition with z-YVAD-fmk (ab141388).

Detecting ferroptosis

Ferroptosis – an iron-dependent form of necrosis – occurs due to production of lipid reactive oxygen species (ROS)11. Cells experiencing ferroptosis show slight morphological features, including mitochondria that are smaller-than-normal and have increased density. One way to confirm the presence of ferroptosis is by measuring lipid peroxides, and by observing whether necrosis is prevented by inhibitors.

 

Key proteins in the ferroptotic pathway

Protein

Function

Role in ferroptosis

GPX4

Lowers lipid hydroperoxides within lipid membranes

Reduced activity in ferroptosis

Glutathione

Substrate for GPX4

Sometimes depleted in ferroptosis, based on the molecular pathway

 

Inhibiting ferroptosis

Chemical inhibitors known to prevent ferroptosis can be used to confirm the presence of ferroptosis. However, knockdown is not an effective technique as ferroptosis is caused by reduced GPX4 activity.

 

Ferroptosis inhibitors and their modes of action

Inhibitor

Mode of action

Ferrostatin-1

Lipid peroxide scavenger

Liproxstatin-1

Unknown. Possibly reduction of free radicals

 

 

Accumulation of lipid peroxides

Ferroptosis depends on the accumulation of ROS. There are several methods at one’s disposal to identify the presence of lipid ROS.

 

Methods to detect the presence of lipid ROS

Assay

Mechanism

How to measure

C11-BODIPY

Detects free radical-induced oxidation

Quantification by flow cytometry

Malondialdehyde quantification

Malondialdehyde is a byproduct of lipid peroxidation

Lipid peroxidation (MDA) assay kit

4-HNE quantification

4-HNE is a byproduct of lipid peroxidation

Antibody-based quantification

 

 

References

  1. Vanden Berghe, T. et al. Determination of apoptotic and necrotic cell death in vitro and in vivo. Methods 61, 117–129 (2013).
  2. Holler, N. et al. Fas triggers an alternative, caspase-8–independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 1, 489–495 (2000).
  3. Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227 (2012).
  4. Cai, Z. et al. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol 16, 55–65 (2014).
  5. Moriwaki, K. & Chan, F. K. M. RIP3: A molecular switch for necrosis and inflammation. Genes Dev 27, 1640–1649 (2013).
  6. Takahashi, N. et al. Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models. Cell Death Dis 3, e437–10 (2012).
  7. Kaiser, W. J. et al. RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition. Proc Natl Acad Sci 111, 7753–7758 (2014).
  8. He, W. et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1[beta] secretion. Cell Res 25, 1285–1298 (2015).
  9. Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015).
  10. Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signaling. Nature 526, 666–671 (2015).
  11. Dixon, S. J. et al. Ferroptosis: an iron-dependent form on nonapoptotic cell death. 149, 1060–1072 (2012).

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Last updated: Jun 6, 2019 at 12:49 PM

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