Zika virus triggers autophagy to exploit host lipid metabolism and drive viral replication.

BACKGROUND: Zika virus (ZIKV), an arbovirus of global concern, has been associated with neurological complications including microcephaly in newborns and Guillain-Barré syndrome in adults. Like other flaviviruses, ZIKV depends on cholesterol to facilitate its replication; thus, cholesterol has been proposed as a therapeutic target to treat the infection using FDA-approved statins. Cholesterol is stored in intracellular lipid droplets (LD) in the form of cholesterol esters and can be regulated by autophagy. We hypothesize that the virus hijacks autophagy machinery as an early step to increase the formation of LD and viral replication, and that interference with this pathway will limit reproduction of virus. METHODS: We pretreated MDCK cells with atorvastatin or other inhibitors of autophagy prior to infection with ZIKV. We measured viral expression by qPCR for NS1 RNA and immunofluorescence for Zika E protein. RESULTS: Autophagy increases in virus-infected cells as early as 6 h post infection (hpi). In the presence of atorvastatin, LD are decreased, and cholesterol is reduced, targeting key steps in viral replication, resulting in suppression of replication of ZIKV is suppressed. Other both early- and late-acting autophagy inhibitors decrease both the number of LD and viral replication. Bafilomycin renders cholesterol is inaccessible to ZIKV. We also confirm previous reports of a bystander effect, in which neighboring uninfected cells have higher LD counts compared to infected cells. CONCLUSIONS: We conclude that atorvastatin and inhibitors of autophagy lead to lower availability of LD, decreasing viral replication. We conclude that bafilomycin A1 inhibits viral expression by blocking cholesterol esterification to form LD. Video Abstract.

Ceramide from sphingomyelin hydrolysis induces neuronal differentiation, whereas de novo ceramide synthesis and sphingomyelin hydrolysis initiate apoptosis after NGF withdrawal in PC12 Cells.

BACKGROUND: Ceramide, important for both neuronal differentiation and dedifferentiation, resides in several membranes, is synthesized in the endoplasmic reticulum, mitochondrial, and nuclear membranes, and can be further processed into glycosphingolipids or sphingomyelin. Ceramide may also be generated by hydrolysis of sphingomyelin by neutral or acidic sphingomyelinases in lysosomes and other membranes. Here we asked whether the differing functions of ceramide derived from different origins. METHODS: We added NGF to PC12 cells and to TrkA cells. These latter overexpress NGF receptors and are partially activated to differentiate, whereas NGF is required for PC12 cells to differentiate. We differentiated synthesis from hydrolysis by the use of appropriate inhibitors. Ceramide and sphingomyelin were measured by radiolabeling. RESULTS: When NGF is added, the kinetics and amounts of ceramide and sphingomyelin indicate that the ceramide comes primarily from hydrolysis but, when hydrolysis is inhibited, can also come from neosynthesis. When NGF is removed, the ceramide comes from both neosynthesis and hydrolysis. CONCLUSION: We conclude that the function of ceramide depends heavily on its intracellular location, and that further understanding of its function will depend on resolving its location during changes of cell status. Video Abstract.

Ceramide and sphingomyelin reportedly are important both for differentiation of nerve cells and for their death. We studied PC12 cells, which can differentiate into neuron-like cells in the presence of nerve growth factor and cells that overexpress receptors for nerve growth factor. By combining various inhibitors, we conclude that in the presence of nerve growth factor ceramide comes from hydrolysis of sphingomyelin, but when nerve growth factor is removed and the cells atrophy and die, sphingomyelin comes from both neosynthesis and hydrolysis.

Assessment of Atg7 and LC3II/LC3, as The Markers of Autophagy, in Sperm of Infertile Men with Globozoospermia: A Case-Control Study.

OBJECTIVE: Assessment of relationship between LC3II/LC3 and Autophagy-related 7 (Atg7) proteins, as markers of autophagy, as well as evaluating the sperm parameters and DNA fragmentation in spermatozoa of infertile men with globozoospermia. MATERIALS AND METHODS: In this case-control study, 10 semen samples from infertile men with globozoospermia and 10 fertile individuals were collected, and the sperm parameters, sperm DNA fragmentation, and main autophagy markers (Atg7 and LC3II/LC3) were assessed according to World Health Organization (WHO) criteria, TUNEL assay, and western blot technique, respectively. RESULTS: The mean of sperm concentration and motility were significantly lower, while the percentage of abnormal spermatozoa and DNA fragmentation were significantly higher in infertile men with globozoospermia compared to fertile individuals (P<0.01). Unlike the relative expression of LC3II/LC3 that did not significantly differ between the two groups, the relative expression of ATG7 was significantly higher in infertile men with globozoospermia compared to fertile individuals (P <0.05). There was a significantly negative correlation between the sperm concentration (r=-0.679; P=0.005) and motility (r=-0.64; P=0.01) with the expression of ATG7, while a significantly positive association was founf between the percentage of DNA fragmentation and expression of ATG7 (0.841; P =0.018). CONCLUSION: The increased expression of ATG7 and unaltered expression of LC3II/LC3 may indicate that the autophagy pathway is initiated but not completely executed in spermatozoa of individuals with globozoospermia. A significant correlation of ATG7 expression with increased sperm DNA fragmentation, reduced sperm concentration, and sperm motility may associate with the activation of a compensatory mechanism for promoting deficient spermatozoa to undergo cell death by the autophagy pathway. Therfore, this pathway could act as a double-edged sword that, at the physiological level, is involved in acrosome biogenesis, while, at the pathological level, such as globozoospermia, could act as a compensatory mechanism.

Higher sensitivity of female cells to ethanol: methylation of DNA lowers Cyp2e1, generating more ROS.

BACKGROUND: Cells taken from mouse embryos before sex differentiation respond to insults according to their chromosomal sex, a difference traceable to differential methylation. We evaluated the mechanism for this difference in the controlled situation of their response to ethanol. METHODS: We evaluated the expression of mRNA for alcohol dehydrogenase (ADH), aldehyde dehyrogenases (ALDH), and a cytochrome P450 isoenzyme (Cyp2e1) in male and female mice, comparing the expressions to toxicity under several experimental conditions evaluating redox and other states. RESULTS: Females are more sensitive to ethanol. Disulfiram, which inhibits alcohol dehydrogenase (ADH), increases cell death in males, eliminating the sex dimorphism. The expressions ADH Class 1 to 4 and ALDH Class 1 and 2 do not differ by sex. However, females express approximately 8X more message for Cyp2e1, an enzyme in the non-canonical pathway. Female cells produce approximately 15% more ROS (reactive oxygen species) than male cells, but male cells contain approximately double the concentration of GSH, a ROS scavenger. Scavenging ROS with N-acetyl cysteine reduces cell death and eliminates sex dimorphism. Finally, since many of the differences in gene expression derive from methylation of DNA, we exposed cells to the methyltransferase inhibitor 5-aza- 2-deoxycytidine; blocking methylation eliminates both the difference in expression of Cyp2e1 and cell death. CONCLUSION: We conclude that the sex-differential cell death caused by ethanol derives from sex dimorphic methylation of Cyp2e1 gene, resulting in generation of more ROS.

Cell death through the ages: The ICDS 25th Anniversary Meeting.

In June of 2019, the International Cell Death Society (ICDS) held its 25th anniversary meeting in New York City at the Icahn School of Medicine at Mount Sinai organized by Drs. Richard A. Lockshin (St. John's University, USA), Zahra Zakeri (Queens College, USA), and Jerry Edward Chipuk (Icahn School of Medicine at Mount Sinai, USA). The three-day event, entitled 'Cell death through the ages: The ICDS 25th anniversary meeting', hosted ninety-one delegates including thirty-four speakers and twenty-two poster presentations. Additionally, the organizers gave special recognition to the twenty-one previous ICDS Lifetime Achievement awardees-those who have significantly contributed to the field of cell death and the growth of the organization. Here, we provide a summary of the meeting and highlight trending research in the fields of cell death, autophagy, immunology, and their impact on health and disease.

Atorvastatin restricts the ability of influenza virus to generate lipid droplets and severely suppresses the replication of the virus.

Influenza virus causes infected cells to generate large numbers of lipid droplets. Because the virus envelope contains substantial cholesterol, we applied atorvastatin (ATV) to Madin-Darby Canine Kidney cells before infecting them. Five micromolars ATV, within physiologic range, strongly (>95%) inhibits reproduction of influenza A as measured by PCR of viral RNA, plaque assay for viable virus, and production of virus nucleoprotein (NP). Inhibition of any of the following can suppress formation of lipid droplets (>-50%) but does not interfere with the production of NP: endoplasmic reticulum stress, autophagy, or production of reactive oxygen substances (ROS). We conclude that, regardless of whether this widely used statin, which is generally considered to be safe, can prevent infection or minimize its severity, inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase pathway to protect against infection by influenza virus or to mitigate its severity warrants further exploration.-Episcopio, D., Aminov, S., Benjamin, S., Germain, G., Datan, E., Landazuri, J., Lockshin, R. A., Zakeri, Z. Atorvastatin restricts the ability of influenza virus to generate lipid droplets and severely suppresses the replication of the virus.

Reduced sperm telomere length in individuals with varicocele is associated with reduced genomic integrity.

Varicocele, defined as enlarged varicose veins in the scrotum, is the most common identifiable cause of male infertility. There are significant correlations between oxidative stress and varicocele-related infertility due to testicular hyperthermia, which can result in low sperm function. In addition, recent excessive oxidative stress can affect sperm telomere length and integrity of sperm DNA. Therefore, we assessed sperm telomere length as a potential marker of paternal genome integrity and leukocyte telomere length as an internal control (real-time PCR), along with sperm chromatin status (TUNEL and chromomycin A3 assay), and lipid peroxidation (Bodipy probe) in 18 infertile men with grade II or III varicocele, and 20 fertile men. Means of sperm parameters, sperm and leukocyte telomere length were significantly lower, while means of sperm DNA fragmentation, protamine deficiency, and lipid peroxidation were significantly higher in infertile men with varicocele compared to fertile men. Therefore, shortened telomere length in sperm and leukocytes is likely associated with increased oxidative stress related to the state of varicocele, which also accounts for increase in sperm DNA fragmentation. Thus, assessment of leukocyte telomere length could be taken as an indicator of antioxidant capacity in an individual, which also affects sperm function.

About canonical, non-canonical and immunogenic cell death: Basic mechanisms and translational applications: A meeting report of the International Cell Death Society.

International Cell Death Society held its 25th meeting, entitled "About canonical, non-canonical, and immunogenic cell death: basic mechanisms and translational applications" in Seoul, South Korea, May 31-June 2, 2018, addressed the most current issues in the field. Now that many types and pathways of cell death are recognized, attention has turned to how the threshold to death is maintained or surpassed, and how and what intracellular signals control the process. Most of the speakers addressed these topics, focusing on mitochondria and on new high-resolution techniques that promise to answer current questions.

Comparison of main molecular markers involved in autophagy and apoptosis pathways between spermatozoa of infertile men with varicocele and fertile individuals.

Abnormal dilatation and tortuosity of the pampiniform plexus within the spermatic cord are termed varicocele which leads to impaired spermatogenesis due to heat-related oxidative stress and cell death. Previously, it was shown that both apoptosis and autophagy pathways were activated by heat in germ cells of mouse in vivo and in vitro. But, status of these pathways is not clear in chronic state of heat stress such as varicocele. Therefore, we aimed to access sperm apoptotic markers (active caspases 3/7 and DNA fragmentation), and autophagic markers (Atg7 and LC3 proteins) as primary outcomes, and also sperm parameters and protamine deficiency as secondary outcomes between 23 infertile men with varicocele and 16 fertile individuals. Sperm parameters were assessed according to World Health Organization 2010 protocol. Apoptotic markers (active caspases 3/7 and DNA fragmentation), autophagic markers (Atg7 and LC3 proteins), and protamine deficiency were evaluated by flow cytometry, fluorescence microscope, and western blotting techniques. Mean of autophagy and apoptosis markers, and also protamine deficiency have significantly increased in infertile men with varicocele compared to fertile individuals, but autophagy and apoptosis markers did not significantly correlate with each other. In conclusion, it seems that both apoptosis and autophagy pathways are independently active in spermatozoa of infertile men with varicocele.

What cell death does in development.

Cell death is prominent in gametogenesis and shapes and sculpts embryos. In non-mammalian embryos one sees little or no cell death prior to the maternal-zygotic transition, but, in mammalian embryos, characteristic deaths of one or two cells occur at the end of compaction and are apparently necessary for the separation of the trophoblast from the inner cell mass. Considerable sculpting of the embryo occurs by cell deaths during organogenesis, and appropriate cell numbers, especially in the CNS and in the immune system, are generated by massive overproduction of cells and selection of a few, with death of the rest. The timing, identity, and genetic control of specific cells that die have been well documented in Caenorhabditis, but in other embryos the stochastic nature of the deaths limit our ability to do more than identify the regions in which cells will die. Complete disruption of the cell death machinery can be lethal, but many mutations of the regulatory machinery yield only modest or no phenotypes, indicating substantial redundancy and compensation of regulatory mechanisms. Most of the deaths are apoptotic and are identified by techniques used to recognize apoptosis, but techniques identifying lysosomes (whether in dying or involuting cells or in the phagocytes that invade the tissue) also reveal patterns of cell death. Aberrant cell deaths that produce known phenotypes are typically localized, indicating that the mechanism of activating a programmed death in a specific region, rather than the mechanism of death, is aberrant. These results lead us to conclude that we need to know much more about the conversations among cells that lead cells to commit suicide.

Regulation of cell survival and death during Flavivirus infections.

Flaviviruses, ss(+) RNA viruses, include many of mankind's most important pathogens. Their pathogenicity derives from their ability to infect many types of cells including neurons, to replicate, and eventually to kill the cells. Flaviviruses can activate tumor necrosis factor α and both intrinsic (Bax-mediated) and extrinsic pathways to apoptosis. Thus they can use many approaches for activating these pathways. Infection can lead to necrosis if viral load is extremely high or to other types of cell death if routes to apoptosis are blocked. Dengue and Japanese Encephalitis Virus can also activate autophagy. In this case the autophagy temporarily spares the infected cell, allowing a longer period of reproduction for the virus, and the autophagy further protects the cell against other stresses such as those caused by reactive oxygen species. Several of the viral proteins have been shown to induce apoptosis or autophagy on their own, independent of the presence of other viral proteins. Given the versatility of these viruses to adapt to and manipulate the metabolism, and thus to control the survival of, the infected cells, we need to understand much better how the specific viral proteins affect the pathways to apoptosis and autophagy. Only in this manner will we be able to minimize the pathology that they cause.

mTOR/p70S6K signaling distinguishes routine, maintenance-level autophagy from autophagic cell death during influenza A infection.

Autophagy, a stress response activated in influenza A virus infection helps the cell avoid apoptosis. However, in the absence of apoptosis infected cells undergo vastly expanded autophagy and nevertheless die in the presence of necrostatin but not of autophagy inhibitors. Combinations of inhibitors indicate that the controls of protective and lethal autophagy are different. Infection that triggers apoptosis also triggers canonical autophagy signaling exhibiting transient PI3K and mTORC1 activity. In terminal autophagy phospho-mTOR(Ser2448) is suppressed while mTORC1, PI3K and mTORC2 activities increase. mTORC1 substrate p70S6K becomes highly phosphorylated while its activity, now regulated by mTORC2, is required for LC3-II formation. Inhibition of mTORC2/p70S6K, unlike that of PI3K/mTORC1, blocks expanded autophagy in the absence of apoptosis but not moderate autophagy. Inhibitors of expanded autophagy limit virus reproduction. Thus expanded, lethal autophagy is activated by a signaling mechanism different from autophagy that helps cells survive toxic or stressful episodes.

Sex-dependent regulation of cytochrome P450 family members Cyp1a1, Cyp2e1, and Cyp7b1 by methylation of DNA.

Sexual differences are only partially attributable to hormones. Cultured male or female cells, even from embryos before sexual differentiation, differ in gene expression and sensitivity to toxins, and these differences persist in isolated primary cells. Male and female cells from Swiss Webster CWF mice manifest sex-distinct patterns of DNA methylation for X-ist and for cytochrome P450 (CYP; family members 1a1, 2e1m, and 7b1. Dnmt3l is differentially expressed but not differentially methylated, and Gapdh is neither differentially methylated nor expressed. CYP family genes differ in expression in whole tissue homogenates and cell cultures, with female Cyp expression 2- to 355-fold higher and Dnmt3l 12- to 32-fold higher in males. DNA methylation in the promoters of these genes is sex dimorphic; reducing methylation differences reduces to 1- to 6-fold differences in the expression of these genes. Stress or estradiol alters both methylation and gene expression. We conclude that different methylation patterns partially explain the sex-based differences in expression of CYP family members and X-ist, which potentially leads to inborn differences between males and females and their different responses to chronic and acute changes. Sex-differential methylation may have medical effects.

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.

Hydrogen peroxide enhances phagocytosis of Pseudomonas aeruginosa in hyperoxia.

Mechanical ventilation with hyperoxia is a necessary treatment for patients with respiratory distress. However, patients on mechanical ventilation have increased susceptibility to infection. Studies including ours have shown that reactive oxygen species (ROS), generated by exposure to prolonged hyperoxia, can cause a decrease in the phagocytic activity of alveolar macrophages. Hydrogen peroxide (H2O2) is a form of ROS generated under hyperoxic conditions. In this study, we examined whether treatment with H2O2 directly affects macrophage phagocytic ability in RAW 264.7 cells that were exposed to either 21% O2 (room air) or 95% O2 (hyperoxia). Moderate concentrations (ranging from 10 to 250 µM) of H2O2 significantly enhanced macrophage phagocytic activity and restored hyperoxia-suppressed phagocytosis through attenuation of hyperoxia-induced disorganization of actin cytoskeleton and actin oxidation. These results indicate that H2O2 at low-moderate concentrations can be beneficial to host immune responses by improving macrophage phagocytic activity.

The execution phase of autophagy associated PCD during insect metamorphosis.

During metamorphosis of Manduca sexta, involution of labial glands follows an autophagic pathway towards programmed cell death (PCD). We looked for evidence of both caspase dependent and independent pathways of PCD by assaying for caspases -1, -2, -3, and -6, proteasomal protease, and cathepsins B & L, using fluorogenic substrates and aldehyde and chloromethylketone inhibitors. The substrates FR-AMC and RR-AMC, preferentially degraded by cathepsins B and L, were the most rapidly degraded, increasing in rate as the gland involuted. Digestion of YVAD-AMC (preferential substrate for caspase-1) and DEVD-AMC (substrate for caspases-3 & -7) was barely detectable, less than 0.02% (on a per-unit-protein basis) of that seen in vertebrate embryos induced to undergo apoptosis. Cleavage of VDVAD-AFC (substrate for caspase -2) and VEID-AFC (substrate for caspase -6) was also assessed, but activity was negligible. Mitochondrial membrane permeabilization (MMP) and cytochrome c release were not detected. Exogenous caspase substrate, polyadenosyl ribose phosphorylase (PARP), is cleaved by labial gland extracts, but only at an acidic pH of 5.5-6.0, and into fragments different from those generated by caspases (confirmed by N-terminal sequencing). The cysteine protease inhibitor leupeptin inhibits PARP cleavage, but the caspase inhibitor DEVD-CHO does not. However, potential caspase-derived fragments of PARP are seen when cytochrome c and dATP are added to cytosolic extracts. Although apoptotic machinery is conserved and functional in this tissue, cell death occurs independently of caspases in metamorphosis. We also postulate that lysosomal proteases play the major proteolytic role similar to the caspase cascade seen in apoptosis.

Activation of cyclin-dependent kinase 5 is a consequence of cell death.

Cyclin-dependent kinase 5 (Cdk5) is similar to other Cdks but is activated during cell differentiation and cell death rather than cell division. Since activation of Cdk5 has been reported in many situations leading to cell death, we attempted to determine if it was required for any form of cell death. We found that Cdk5 is activated during apoptotic deaths and that the activation can be detected even when the cells continue to secondary necrosis. This activation can occur in the absence of Bim, calpain, or neutral cathepsins. The kinase is typically activated by p25, derived from p35 by calpain-mediated cleavage, but inhibition of calpain does not affect cell death or the activation of Cdk5. Likewise, RNAi-forced suppression of the synthesis of Cdk5 does not affect the incidence or kinetics of cell death. We conclude that Cdk5 is activated as a consequence of metabolic changes that are common to many forms of cell death. Thus its activation suggests processes during cell death that will be interesting or important to understand, but activation of Cdk5 is not necessary for cells to die.

Detection of apoptosis in mammalian development.

Mammalian development is dependent on an intricate orchestration of cell proliferation and death. Deregulation in the levels, localization, and type of cell death can lead to disease and even death of the developing embryo. The mechanisms involved in such deregulation are many; alterations and or manipulations of these can aid in the detection, prevention and possible treatments of any effects this de-regulation may have. Here we describe how cell death can be detected during mammalian development, using diverse staining and microscopy methods, while taking advantage of the advancements in cell death mechanisms, derived from biochemical and teratological studies in the field.