At the cellular level, all higher organisms undergo apoptosis. This tightly regulated process, whereby cells self-destruct for the greater good of the being, is fundamental to organismal development, health maintenance, and disease prevention. “[Apoptosis] allows for the renewal of other cells and the control of abnormal cells grown within the body. This is important for embryonic development and maintaining tissue homeostasis within bodily systems such as the immune system,” explains Matthew Miceli, global product manager, research reagents, at BD.
During viral infections, for example, apoptosis kills off invaded cells to hinder the spread of virus, sparing the whole organism. In neurobiology, this same phenomenon helps to prune synaptic connections, strengthening pathways that are used frequently, and disposing of the rarely used ones. As with most homeostatic mechanisms in the body, when apoptosis fails to work properly, disease arises, for example, in the form of neurodegeneration or cancer.
“In the fields of immunology, cancer, developmental biology, and drug discovery, identifying and investigating cells undergoing apoptosis is essential,” says Miceli. Ken Lau, technical marketing manager for BioLegend, concurs and adds “Understanding the nature of how a cell dies can lead to important revelations on how we might preserve its lifespan.”
Apoptosis occurs when cells receive either an internal or external signal that triggers the release of proteases and eventual cascade of carefully orchestrated, tightly controlled events causing cellular ablation. Morphologically, the cell will shrink and disengage from its neighbor. The cell surface will bleb or bubble as fragments break away. DNA within the nucleus condenses tightly and breaks into uniformly sized fragments. The nucleus disintegrates soon after, followed by the rest of the cell. A clean-up crew of phagocytic cells then disposes of the dead cell and debris.
Hardy Rideout, collaborating scientist at the Laboratory of Neurodegenerative Diseases Biomedical Research Foundation at Academy of Athens, studies neuronal cell death in models of Parkinson’s disease, specifically, those linked to mutations in the gene encoding LRRK2. He explains, “Clearly defining the death signaling cascade triggered by mutant LRRK2 allows the identification of novel therapeutic targets that can serve as the basis for rational drug design.” His group’s most recent work describes apoptotic pathways triggered by mutant versions of LRRK2.
Monitoring apoptosis
The events that occur in the cell vary depending on the apoptotic pathway. The intrinsic pathway is mediated by the mitochondria following internal triggers such as DNA damage, while the extrinsic pathway is controlled by extracellular death receptors in response to external provocation such as growth factor withdrawal.
Activation of the intrinsic pathway leads to initiation of Bcl-2 family signaling cascade, mitochondrial membrane depolarization, and subsequent leakage of cytochrome c and APAF-1 into the cytosol. These proteins form the apoptosome along with caspase 9, which triggers effector caspase (caspases-3/-7) activation and concomitant exposure of phosphatidylserine (PS) to the extracellular side of the cell membrane.
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The extrinsic pathway is activated following binding of ligands to members of the tumor necrosis factor family ultimately leading to caspase-8 activation. As with the intrinsic pathway, effector caspases are activated and PS is exposed extracellularly.
From this point on, the two pathways behave the same. At this late-stage in apoptosis, DNA fragments and the cell membrane blebs. Morphologically, the cell shrinks and phagocytosis ensues.
Within a population of cells, “different cells may be in different stages of apoptosis at the same time point,” says Miceli. “It’s best to screen for early and later processes in initial experiments and then select an assay based on the sensitivity of the results for that cell type.”
Rideout believes that for post-mitotic cells such as neurons, the gold standard for identifying degenerating cells is nuclear morphology, which appears as condensed fragmented chromatin. “[These observations] can be buttressed by employing stains for late-stage apoptotic markers, like activated caspase-3. Careful examination of nuclear morphology with DAPI coupled with other early classic apoptotic markers (such as cytochrome c) can help stage dying cells at earlier points in the pathway.”
According to Mohammed Mamunur Rahman, business development and tech support manager at MBL International, there are a variety of ways to assess apoptosis. These include using Annexin V to stain for exposed PS on the cell surface, DNA electrophoresis to monitor fragmentation, electron microscopy to observe canonical apoptotic morphological changes of the cell membrane and nucleus, and apoptosis-associated caspase activation assays to examine substrate cleave events in vitro.
Lau says that early events, such as mitochondrial membrane depolarization, can be detected with BioLegend’s MitoSpy™. Later events can be monitored with DNA-binding dyes, such as BioLegend’s Helix NP™and propidium iodide. “Having multiple points of confirmation makes it easier to determine that the cell has committed to apoptosis,” he adds.
BD also offers an extensive selection of products to study different aspects of apoptosis. “We have a wide variety of fluorochrome choices for Annexin V that allows for flexibility when incorporating into multicolor panels,” explains Miceli. Additionally, MitoScreen kits and Mitostatus dyes detect mitochondrial changes at the single cell level. For caspase activity, Live Caspase Probes, anti-caspase-3 antibody, and anti-cleaved PARP antibodies may be useful. And for DNA fragmentation, BD has APO-DIRECT and APO-BRDU kits that label DNA breaks for flow cytometric analysis.
If single-cell assessment is not required, Gary Kasof, senior fellow, development, at Cell Signaling Technology (CST), advises using Western blot for cleaved caspases, apoptotic family members, and caspase substrate antibodies. He says that the techniques used will depend on the type of sample. For example, if looking at blood, scientists may want to combine a flow cytometry assay, such as Annexin V, with fluorescently conjugated cleaved caspase/substrate antibodies. If examining tissue, then perhaps choose TUNEL along with IHC-based caspase activation. Live cell observation, on the other hand, may call for evaluating the effects of caspase inhibitors.
CST offers thoroughly validated antibodies to probe different cell death pathways depending on the final readout (for example, Western blot, immunohistochemistry, immunofluorescence, etc.). Kasof often recommends CST’s antibody sampler kits as a starting point. “We have curated the antibody sampler kits to take some of the questions out of which targets to look at when monitoring apoptosis.”
It’s also important to keep in mind that not all cell types display all the classical features of apoptosis and vary in their response to apoptotic triggers.
Image: Confocal immunofluorescent analysis of E14 mouse digits using Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin(red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
It’s also important to keep in mind that not all cell types display all the classical features of apoptosis and vary in their response to apoptotic triggers. “Many cell lines that we use are not necessarily sensitive to all death stimuli. Over-expressing mutant forms of LRRK2 in primary neurons can lead to apoptotic cell death, but this is not the case for other cell lines we use to study LRRK2 function. HEK293T cells, for example, are resistant to mutant LRRK2 induced death,” says Rideout of his research.
Apoptosis versus necrosis
“Morphologically, apoptosis is distinct from necrosis. Apoptosis is characterized by cell shrinkage, membrane blebbing, DNA laddering, nuclear condensation, and phagocytic engulfment; whereas necrosis is characterized by cell swelling and lysis,” says Kasof.
It is essential to distinguish between these pathways since there are different consequences to the surrounding cells in terms of inflammatory responses. Additionally, Kasof says that there are therapeutic drugs that target these pathways.
As an example, he says that in cancer cells, apoptotic resistance markers are upregulated and that they may actually be more vulnerable to necroptosis (a programmed version of necrosis). “As a result, intents to kill these cells by traditional apoptotic stimuli, may unintentionally lead to an inflammatory process of necrosis.”
Rahman recommends MBL’s MEBCYTO Apoptosis Kit, which contains Annexin V-FITC and propidium iodide (PI) to help distinguish between necrosis and apoptosis. Annexin V will stain both apoptotic and necrotic cells. However, PI will only stain necrotic cells.
Even this assay, however, must be used with caution. “It may be hard to distinguish late apoptotic cells that become PI positive,” says Kasof. And he has a list of other things to consider: It was recently reported that cells about to undergo necroptosis can become PI negative before the loss of membrane integrity, non-apoptotic activities for caspases were recently described, and TUNEL assays may be misleading since DNA damage, which is a late-stage event, is found in both apoptosis and necrosis. All of these findings are things to keep in mind when examining death pathways in cells.
But Rideout says that the field has advanced considerably in recent years, and that is encouraging. Despite the long checklists of things that need to be considered when carrying out these experiments, scientists have more tools available than ever before to properly assess apoptosis, tease out signaling pathway, and unlock the mysteries underlying disease and development.
Image (top) courtesy of Nephron, [CC BY-SA 3.0], via Wikimedia Commons.