Anna-Mart Engelbrecht

The variability of autophagy and cell death susceptibility: Unanswered questions

Impaired autophagic machinery is implicated in a number of diseases such as heart disease, neurodegeneration and cancer. A common denominator in these pathologies is a dysregulation of autophagy that has been linked to a change in susceptibility to cell death. Although we have progressed in understanding the molecular machinery and regulation of the autophagic pathway, many unanswered questions remain. How does the metabolic contribution of autophagy connect with the cell’s history and how does its current autophagic flux affect metabolic status and susceptibility to undergo cell death? How does autophagic flux operate to switch metabolic direction and what are the underlying mechanisms in metabolite and energetic sensing, metabolite substrate provision and metabolic integration during the cellular stress response? In this article we focus on unresolved questions that address issues around the role of autophagy in sensing the energetic environment and its role in actively generating metabolite substrates. We attempt to provide answers by explaining how and when a change in autophagic pathway activity such as primary stress response is able to affect cell viability and when not. By addressing the dynamic metabolic relationship between autophagy, apoptosis and necrosis we provide a new perspective on the parameters that connect autophagic activity, severity of injury and cellular history in a logical manner. Last, by evaluating the cell’s condition and autophagic activity in a clear context of regulatory parameters in the intra- and extracellular environment, this review provides new concepts that set autophagy into an energetic feedback loop, that may assist in our understanding of autophagy in maintaining healthy cells or when it controls the threshold between cell death and cell survival.

p38 and JNK have distinct regulatory functions on the development of apoptosis during simulated ischaemia and reperfusion in neonatal cardiomyocytes

Abstract. Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (SI/R) were used as an in vitro model to delineate the role(s) of extracellular signal–regulated kinase (ERK), p38 and c–Jun NH2–terminal protein kinase (JNK), as well as PKB in apoptosis. Exposure of the myocytes to SI (simulated ischaemia – energy depletion induced by KCN and 2-deoxy-D–glucose) reduced cell viability, as measured by the 3–[4,5–dimethylthiazol–2–yl]–2,5–diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis as evidenced by caspase–3 activation and poly(ADP–ribose) polymerase (PARP) cleavage. However, morphological evidence of increased apoptosis, detected by staining with Hoechst 33342, was only seen in response to reperfusion. This suggests that although ischaemic conditions are sufficient to induce cellular markers of apoptosis (PARP cleavage and caspase–3 activation), reperfusion is required to complete the apoptotic pathway in these cells. Furthermore, SI resulted in a rapid, strong, biphasic activation of p38 concomitant with a weak and transient activation of the two ERK isoenzymes, p42/p44–MAPK. Reperfusion for 5 minutes resulted in a strong phosphorylation of p42/p44–MAPK, while no additional p38 activation was seen at this stage. On the other hand, p46/p54–MAPK (JNK) was phosphorylated in response to 5 minutes of reperfusion only and not during SI alone. A peak of PKB/Akt (Ser473) activity was seen within 5 minutes of exposure to SI, whereas PKB/Akt (Thr308) phosphorylation remained at the baseline level. Both PKB/Akt phosphorylation sites (Ser473 and Thr308) were phosphorylated after 5 minutes of reperfusion. Inhibition of PI–3–kinase activity, using wortmannin, decreased phosphorylation on both sites during SI. However, only SI/R-induced PKB/Akt phosphorylation on Thr308 was reduced by wortmannin. Myocytes pre–treated with SB203580, a p38–inhibitor, displayed a significant increase in cell viability [63.67 ± 1.85 to 84.33 ± 4.8% (p < 0.05)] and attenuation of the apoptotic index during SI/R [22.6 ± 2.94% to 9 ± 0.43% (p < 0.001)], while SP600125, a specific JNK inhibitor, caused a significant increase in caspase–3 activation [1.66 ± 0.03 fold to 2.56 ± 0.27 fold (p < 0.001)] and apoptotic index [22.6 ± 2.94% to 32.75 ± 6.13% (p < 0.05)]. However, PD98059, an ERK inhibitor, failed to affect apoptosis during SI/R. Inhibition of PI–3–kinase prevented the increase in mitochondrial viability usually observed during reperfusion. Interestingly, wortmannin caused a significant increase in PARP cleavage during reperfusion, but had no effect on caspase–3 activation or the apoptotic index. Our results suggest that p38 has a pro–apoptotic role while JNK phosphorylation is protective in our cell model and that these kinases act via caspase–3 to prevent or promote cell survival in response to SI/R–induced injury.

Diet-induced obesity alters signalling pathways and induces atrophy and apoptosis in skeletal muscle in a prediabetic rat model.

Pro‐inflammatory and stress‐activated signalling pathways are important role players in the pathogenesis of obesity and insulin resistance. Obesity and type II diabetes are associated with chronic, low‐grade inflammation and elevated tumour necrosis factor‐α (TNF‐α) levels. There is increasing evidence that TNF‐α may play a critical role in skeletal muscle atrophy. However, the effects of obesity‐induced insulin resistance on these signalling pathways are poorly understood in skeletal muscle. Therefore, the present study addressed the effects of obesity‐induced insulin resistance on the activity of the ubiquitin ligases, nuclear factor‐κB, p38 MAPK and phosphoinositide 3‐kinase signalling pathways in the gastrocnemius muscle and compared these with muscle of standard chow‐fed control rats. Male Wistar rats were randomly allocated to a control diet group (standard commercial chow; 60% carbohydrates, 30% protein and 10% fat) or a cafeteria diet group (65% carbohydrates, 19% protein and 16% fat) for 16 weeks. Blood analysis was conducted to determine the impact of the model of obesity on circulating insulin, glucose, free fatty acids, TNF‐α and angiotensin II concentrations. The experimental animals were 18% heavier and had 68% greater visceral fat mass than their control counterparts and were dyslipidaemic. Significant increases in the ubiquitin ligase and MuRF‐1, as well as in caspase‐3 and poly‐ADP‐ribose polymerase cleavage were observed in the muscle of obese animals compared with the control rats. We propose that dyslipidaemia may be a mechanism for the activation of inflammatory/stress‐activated signalling pathways in obesity and type II diabetes, which will lead to apoptosis and atrophy in skeletal muscle.

Autophagy upregulation promotes survival and attenuates doxorubicin-induced cardiotoxicity

This study evaluated whether the manipulation of autophagy could attenuate the cardiotoxic effects of doxorubicin (DXR) in vitro as well as in a tumour-bearing mouse model of acute doxorubicin-induced cardiotoxicity. We examined the effect of an increase or inhibition of autophagy in combination with DXR on apoptosis, reactive oxygen species (ROS) production and mitochondrial function. H9C2 rat cardiac myoblasts were pre-treated with bafilomycin A1 (autophagy inhibitor, 10 nM) or rapamycin (autophagy inducer, 50 μM) followed by DXR treatment (3 μM). The augmentation of autophagy with rapamycin in the presence of DXR substantially ameliorated the detrimental effects induced by DXR. This combination treatment demonstrated improved cell viability, decreased apoptosis and ROS production and enhanced mitochondrial function. To corroborate these findings, GFP-LC3 mice were inoculated with a mouse breast cancer cell line (EO771). Following the appearance of tumours, animals were either treated with one injection of rapamycin (4 mg/kg) followed by two injections of DXR (10 mg/kg). Mice were then sacrificed and their hearts rapidly excised and utilized for biochemical and histological analyses. The combination treatment, rather than the combinants alone, conferred a cardioprotective effect. These hearts expressed down-regulation of the pro-apoptotic protein caspase-3 and cardiomyocyte cross-sectional area was preserved. These results strongly indicate that the co-treatment strategy with rapamycin can attenuate the cardiotoxic effects of DXR in a tumour-bearing mouse model.

Cell death: a dynamic response concept.

Autophagy, apoptosis and necrosis have previously been described as distinct static processes that induce and execute cell death. Due to an increased use of novel techniques in mapping cellular death; techniques which allow for reporting of real-time data; the existence of “grey zones” between cell death modes and the existence of the “point of no return” within these have been revealed. This revelation demands for the integration of new concepts in describing the cellular death process. Furthermore, since the contribution of autophagy in cell death or cell survival is still poorly understood, it is important to accurately describe its function within the dynamic framework of cell death. In this review cell death is viewed as a dynamic and integrative cellular response to ensure the highest likelihood of self preservation. Suggestions are offered for conceptualizing cell death modes and their morphological features, both individually and in relation to one another. It addresses the need for distinguishing between dying cells and dead cells so as to better locate and control the onset of cell death. Most importantly, the fundamental role of autophagy, autophagic flux, and the effects of the intracellular metabolic environment on the kinetics of the cell death modes are stressed. It also contextualizes the kinetic dimension of cell death as a process and aims to contribute towards a better understanding of autophagy as a key mechanism within this process. Understanding the dynamic nature of the cell death process and autophagy’s central role can reveal new insight for therapeutic intervention in preventing cell death.

Docosahexaenoic acid induces apoptosis in colorectal carcinoma cells by modulating the PI3 kinase and p38 MAPK pathways.

Numerous studies have shown that long-chain polyunsaturated fatty acids can kill cancer cells in vitro as well as in vivo, while normal cells remain unaffected. Unfortunately, the cellular and molecular mechanisms responsible for this phenomenon are still poorly understood. The aim of this study was to investigate the potential chemopreventative/antiproliferative potential of docosahexaenoic acid (DHA) in an adenocarcinoma cell line (CaCo2 cells) and to evaluate the signalling pathways modulated by it. DHA (5-50 microM) significantly inhibited cell viability in a dose-dependent manner in CaCo2 cells, while the viability of normal colon cells (NCM460 cells) was not compromised. DHA also induced apoptosis in CaCo2 cells, as indicated by increases in caspase-3 activation and poly-ADP-ribose polymerase cleavage. Signalling proteins, which include extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), Akt and p53 were analysed by Western blotting using phosphospecific and total antibodies. The protein inhibitors wortmannin (phosphoinositide 3 kinase inhibitor), PD 98059 (MEK inhibitor) and SB 203580 (p38 inhibitor) as well as silencing RNA [small interfering RNA (siRNA)] of the p38 MAPK protein, were used to investigate cross-talk between signalling pathways. DHA supplementation significantly suppressed Akt phosphorylation, which also correlated with decreased cell viability and increased apoptosis in CaCo2 cells. Furthermore, siRNA experiments suggested a possible role for p38 MAPK in the phosphorylation of p53 at Ser15, a site which is associated with DNA damage. DHA might thus exert its beneficial effects by means of increased apoptosis and suppression of the important survival-related kinase, Akt.

At the core of survival: Autophagy delays the onset of both apoptotic and necrotic cell death in a model of ischemic cell injury

Ischemic cell injury leads to cell death. Three main morphologies have been described: apoptosis, cell death with autophagy and necrosis. Their inherent dynamic nature, a point of no return (PONR) and molecular overlap have been stressed. The relationship between a defined cell death type and the severity of injury remains unclear. The functional role of autophagy and its effects on cell death onset is largely unknown. In this study we report a differential induction of cell death, which is dependent on the severity and duration of an ischemic insult. We show that mild ischemia leads to the induction of autophagy and apoptosis, while moderate or severe ischemia induces both apoptotic and necrotic cell death without increased autophagy. The autophagic response during mild injury was associated with an ATP surge. Real-time imaging and Fluorescence Resonance Energy Transfer (FRET) revealed that increased autophagy delays the PONR of both apoptosis and necrosis significantly. Blocking autophagy shifted PONR to an earlier point in time. Our results suggest that autophagic activity directly alters intracellular metabolic parameters, responsible for maintaining mitochondrial membrane potential and cellular membrane integrity. A similar treatment also improved functional recovery in the perfused rat heart. Taken together, we demonstrate a novel finding: autophagy is implicated only in mild injury and positions the PONR in cell death.

Mitochondrial catastrophe during doxorubicin‐induced cardiotoxicity: a review of the protective role of melatonin

Anthracyclines, such as doxorubicin, are among the most valuable treatments for various cancers, but their clinical use is limited due to detrimental side effects such as cardiotoxicity. Doxorubicin‐induced cardiotoxicity is emerging as a critical issue among cancer survivors and is an area of much significance to the field of cardio‐oncology. Abnormalities in mitochondrial functions such as defects in the respiratory chain, decreased adenosine triphosphate production, mitochondrial DNA damage, modulation of mitochondrial sirtuin activity and free radical formation have all been suggested as the primary causative factors in the pathogenesis of doxorubicin‐induced cardiotoxicity. Melatonin is a potent antioxidant, is nontoxic, and has been shown to influence mitochondrial homeostasis and function. Although a number of studies support the mitochondrial protective role of melatonin, the exact mechanisms by which melatonin confers mitochondrial protection in the context of doxorubicin‐induced cardiotoxicity remain to be elucidated. This review focuses on the role of melatonin on doxorubicin‐induced bioenergetic failure, free radical generation, and cell death. A further aim is to highlight other mitochondrial parameters such as mitophagy, autophagy, mitochondrial fission and fusion, and mitochondrial sirtuin activity, which lack evidence to support the role of melatonin in the context of cardiotoxicity.

Tumor necrosis factor alpha (TNF-α) inactivates the PI3-kinase/PKB pathway and induces atrophy and apoptosis in L6 myotubes.

Muscle atrophy poses a serious concern to patients inflicted with inflammatory diseases. An increasing body of evidence implies that TNF-α plays a critical role in muscle atrophy in a number of these clinical settings. The mechanisms mediating its effects are not completely understood and conflicting data regarding its anabolic and catabolic actions exists. To examine the functional significance and detailed morphological characteristics of TNF-α-induced muscle proteolysis, differentiated L6 myotubes were subjected to increasing concentrations of recombinant TNF-α for 24 and 48 h. Data analysis of cell death showed that TNF-α induced a combination of apoptosis and necrosis in high concentrations. TNF-R1, rather than TNF-R2, was significantly upregulated. In addition, the transcription factors, NF-κB and FKHR were rapidly activated thus leading to increased expression of ubiquitin ligases, MuRF-1 and MAFbx. Muscle fiber diameter decreased with increasing TNF-α concentrations and was associated with attenuation of the PI3-K/Akt pathway as well as significant reductions in differentiation markers. Furthermore, treatment of L6 myotubes with exogenous TNF-α strongly potentiates its proteolytic effects through certain MAPKs that are activated. These observations suggest that TNF-α induces muscle proteolysis in a dose-dependent manner via various signal transduction pathways.

AHNAK: The giant jack of all trades

The nucleoprotein AHNAK is an unusual and somewhat mysterious scaffolding protein characterised by its large size of approximately 700 kDa. Several aspects of this protein remain uncertain, including its exact molecular function and regulation on both the gene and protein levels. Various studies have attempted to annotate AHNAK and, notably, protein interaction and expression analyses have contributed greatly to our current understanding of the protein. The implicated biological processes are, however, very diverse, ranging from a role in the formation of the blood-brain barrier, cell architecture and migration, to the regulation of cardiac calcium channels and muscle membrane repair. In addition, recent evidence suggests that AHNAK might be yet another accomplice in the development of tumour metastasis. This review will discuss the different functional roles of AHNAK, highlighting recent advancements that have added foundation to the proposed roles while identifying ties between them. Implications for related fields of research are noted and suggestions for future research that will assist in unravelling the function of AHNAK are offered.