Dear Editor,

Having read with great interest the article “Ethyl pyruvate protects PC12 cells from oxygen-glucose deprivation: A potential role in ischemic cerebrovascular disease.” published in your journal [1], we were compelled to reflect on cell death pathways in the PC12 "energy" deprivation models and share with your readers these thoughts.
In short, the paper by Li et al, demonstrated the beneficial effects of Ethyl Pyruvate (EP) in an in vitro PC12 cell line Oxygen Glucose Deprivation (OGD) model (2 h of OGD in a no glucose medium under 8% O2 atmosphere, followed by high glucose medium complemented with 5mM EP incubated in normoxia conditions)”. This was followed by an in vivo approach utilizing a middle cerebral artery occlusion model of ischemia in rats and subsequent treatment with EP.
The authors conclude that in vitro EP treatment increases cell viability after OGD, reduces the percentage of OGD-attributed apoptosis and increases the OGD-decreased levels of neurotrophic factors (BDNF, NGF), while in the in vivo model EP decreases infract volume and improves neurobehavioral parameters. The comments herein regard the in vitro part of the experimentation from which an apoptosis-depended death was proposed as a result of OGD exposure, based on data procured utilizing TUNEL apoptotic assay, the Annexin-V-FITC/PI cell cytometry assay and relative quantification of molecular markers such as Bax, Bcl-2, NF-κB, p65, NCID and Notch-1 using qPCR and Western blotting.
PC12 cell line, being of neuronal origin, is a widely accepted model cell line for research pertaining to neuronal function at the cellular level. In particular, it is used for the elucidation of the mechanisms involved in cell death or survival in different pathological modalities including cerebral or spinal cord ischemia-hypoxia and traumatic brain injury.
At the cellular level, ischemia-hypoxia is simulated by culturing PC12 cells in low oxygen concentration, in varying combinations of nutrient and serum deprivation, with or without oxygen reperfusion and for varying time intervals [1–3]. Pertaining to traumatic brain injury (TBI), involvement of the glutamatergic transmission and excitotoxic phenomena is also investigated [4].
In the past 30 years a great deal or research has been conducted in an effort to understand the processes of cell death. This lead to the clarification of different processes and pathways involved, with apoptosis being the major and best understood example of programmed cell death. “Apoptosis can be triggered by a variety of stimuli and employs a wide array of signaling pathways to exert its effects”. DNA damage is the best understood example of a stimulus leading to cell death, through the p53-dependent pathway. Of course, there are examples of physiological apoptotic cell death, such as those occurring during human embryo development or even aging [5].
Soon necrosis came into play as an "ordered" process of "chaotic" cell death in the sense that ion imbalance leads to gradients collapse, cell swelling and finally, cell death. This type of death is examined in neurodenegeration studies employing PC12 cells as a model cell line. It relates to cerebral ischemia and TBI, in respect that elevated extracellular concentration of glutamate triggers excitotoxicity by augmenting intracellular calcium concentration in the postsynaptic neurons, thus initiating the processes of necrosis, through calpain proteases activation [6].
Both processes recruit intracellular "executioners" in order to infict cell death The apoptotic “executioners” are caspases (cysteine aspartate specific proteases), while necrosis recruits calpains (calcium-dependent, non-lysosomal cysteine proteases) [4,5].
For some years now autophagy, a process in which a cell consumes itself usually as a coping mechanism under adverse low energy environments such as oxygen and nutrient deprivation, turns out to be an able contestant of the above cell death processes leading inadvertently to cell death should it is allowed to proceed long enough. There are three types of autophagy for cytoplasmic material and two types of mitophagy, specific for mitochondrial degradation. All of the above, ultimately utilize lysosomal cathepsin proteases to degrade the autophagosome engulfed cytoplasmic material [6].

Reflecting on the above, cell death “pathways” are complex processes initiated by a wide variety of stimuli that in order to allow for cell death, need to act long enough to surpass all checkpoints that would ensure cell survival.Cell death in all cases is brought about by activated of at least one of three different classes of proteases (caspases, calpains, cathepsins). In addition, a cross talk of apoptosis, necrosis and autophagy has been demonstrated, putting forth the importance of the interplay between those mechanisms that will lead to the final cellular “decision” of the mode of cell death (Fig.1) [7,8].

1. Download high-res image (298KB)
2. Download full-size image

Fig. 1. Interplay of the three main mediators of cell death (Caspases, Calpains, Cathepsins) in cell death processes.
Following an injurious stimuli the cell may undergo apoptosis, necrosis or autophagy, a decision regulated by the cross-talk of caspases, calpains and cathepsins. Activated caspases can cleave autophagy mediators such as Beclin-1 and deactivate it. Alternatively caspase mediated cleavage of Atg4G can sensitize cell to autophagy. Autophagy activation can inhibit caspase activation shutting off apoptosis. Caspases can also have an effect on calpains, activating them via cleavage promoting, either directly or indirectly through the lysosomal cathepsins, necrosis. Calpains except from their central role in necrosis can modulate apoptosis by activating or deactivating caspases.

In a similar article to the one published in this journal we evaluated the degree of involvement of each of the above-mentioned classes of proteases in a PC12 hypoxia model, utilizing similar but not identical conditions. We showed that inhibition of cathepsin D and E proteases could promote the survival of PC12 cells cultivated under oxygen and glucose deprivation conditions for 16 h [2,3]. In 2016 using our PC12 hypoxia model we showed that oxygen-glucose deprivation modulates the unfolded protein response and inflicts autophagy in the PC12 cells [9].
Having in mind the complexity of the cell death pathways and our divergent observations in similar PC12 hypoxia “energy” depletion models, I am compelled to propose that investigation in this vital area needs to address not only the activation of one cell death pathway but the concomitant idleness of all the others. For example, there are many markers that can be used to elucidate the degree of involvement of autophagic processes and monitoring autophagic flux such as conversion of LC3-I to LC3-II, Beclin-1 and Atg-5, all hallmarks of autophagic initiation and progression [10]. Similarly, calpain activation assessment can also be employed in order to assess for calpain mediated necrotic cell death [7].
This way conclusions can be drawn in a safer way and provide concrete evidence to the very intriguing and complex situation that arises from oxygen and energy deprivation in the nervous system. Furthermore, light can be shed on if and how cytoprotective agents such as PE used in the original article modulate the apoptotic-necrotic-autophagic interplay, allowing the development of more fine-tuned and efficient pharmacological agents for clinical practice.

References

[1]

W. Li, J. Lou, L. Wei, H. Bai, Y. Zhang, Y. HeEthyl pyruvate protects PC12 cells from oxygen-glucose deprivation: a potential role in ischemic cerebrovascular disease

Biomed. Pharmacother., 92 (2017), pp. 168-174

ArticleDownload PDFView Record in Scopus

[2]

A. Kritis, C. Pourzitaki, I. Klagas, M. Chourdakis, M. AlbaniProteases inhibition assessment on PC12 and NGF treated cells after oxygen and glucose deprivation reveals a distinct role for aspartyl proteases

PLoS One, 6 (10) (2011), p. e25950

CrossRef

[3]

S.A. Raya, V. Trembovler, E. Shohami, P. LazaroviciA tissue-culture ischemic device to study eicosanoid release by pheochromocytoma Pc12 cultures

J. Neurosci. Methods, 50 (2) (1993), pp. 197-203

ArticleDownload PDFView Record in Scopus

[4]

A.A. Kritis, E.G. Stamoula, K.A. Paniskaki, T.D. VavilisResearching glutamate - induced cytotoxicity in different cell lines: a comparative/collective analysis/study

Front. Cell. Neurosci., 9 (2015), p. 91

[5]

S. ElmoreApoptosis: a review of programmed cell death

Toxicol. Pathol., 35 (4) (2007), pp. 495-516

CrossRefView Record in Scopus

[6]

K.R. Parzych, D.J. KlionskyAn overview of autophagy: morphology, mechanism, and regulation

Antioxid. Redox. Signal., 20 (3) (2014), pp. 460-473

CrossRefView Record in Scopus

[7]

V. Nikoletopoulou, M. Markaki, K. Palikaras, N. TavernarakisCrosstalk between apoptosis, necrosis and autophagy

Biochim. Biophys. Acta, 1833 (12) (2013), pp. 3448-3459

ArticleDownload PDFView Record in Scopus

[8]

M. Artal-Sanz, N. TavernarakisProteolytic mechanisms in necrotic cell death and neurodegeneration

FEBS Lett., 579 (15) (2005), pp. 3287-3296

ArticleDownload PDFCrossRefView Record in Scopus

[9]

T. Vavilis, N. Delivanoglou, E. Aggelidou, E. Stamoula, K. Mellidis, A. Kaidoglou, A. Cheva, C. Pourzitaki, K. Chatzimeletiou, A. Lazou, M. Albani, A. KritisOxygen-glucose deprivation (OGD) modulates the unfolded protein response (UPR) and inflicts autophagy in a PC12 hypoxia cell line model

Cell. Mol. Neurobiol., 36 (5) (2016), pp. 701-712

CrossRefView Record in Scopus

[10]

B.E. Fitzwalter, A. ThorburnRecent insights into cell death and autophagy

FEBS J., 282 (22) (2015), pp. 4279-4288

CrossRefView Record in Scopus