Mehrdad Alirezaei

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Disruption of autophagy--a key homeostatic process in which cytosolic components are degraded and recycled through lysosomes--can cause neurodegeneration in tissue culture and in vivo. Upregulation of this pathway may be neuroprotective, and much effort is being invested in developing drugs that cross the blood brain barrier and increase neuronal autophagy. One well-recognized way of inducing autophagy is by food restriction, which upregulates autophagy in many organs including the liver; but current dogma holds that the brain escapes this effect, perhaps because it is a metabolically privileged site. Here, we have re-evaluated this tenet using a novel approach that allows us to detect, enumerate and characterize autophagosomes in vivo. We first validate the approach by showing that it allows the identification and characterization of autophagosomes in the livers of food-restricted mice. We use the method to identify constitutive autophagosomes in cortical neurons and Purkinje cells, and we show that short-term fasting leads to a dramatic upregulation in neuronal autophagy. The increased neuronal autophagy is revealed by changes in autophagosome abundance and characteristics, and by diminished neuronal mTOR activity in vivo, demonstrated by a reduction in levels of phosphorylated S6 ribosomal protein in Purkinje cells. The increased abundance of autophagosomes in Purkinje cells was confirmed using transmission electron microscopy. Our data lead us to speculate that sporadic fasting might represent a simple, safe and inexpensive means to promote this potentially therapeutic neuronal response.

Autophagy can play an important part in protecting host cells during virus infection, and several viruses have developed strategies by which to evade or even exploit this homeostatic pathway. Tissue culture studies have shown that poliovirus, an enterovirus, modulates autophagy. Herein, we report on in vivo studies that evaluate the effects on autophagy of coxsackievirus B3 (CVB3). We show that in pancreatic acinar cells, CVB3 induces the formation of abundant small autophagy-like vesicles and permits amphisome formation. However, the virus markedly, albeit incompletely, limits the fusion of autophagosomes (and/or amphisomes) with lysosomes, and, perhaps as a result, very large autophagy-related structures are formed within infected cells; we term these structures megaphagosomes. Ultrastructural analyses confirmed that double-membraned autophagy-like vesicles were present in infected pancreatic tissue and that the megaphagosomes were related to the autophagy pathway; they also revealed a highly organized lattice, the individual components of which are of a size consistent with CVB RNA polymerase; we suggest that this may represent a coxsackievirus replication complex. Thus, these in vivo studies demonstrate that CVB3 infection dramatically modifies autophagy in infected pancreatic acinar cells.

There is compelling evidence to support the idea that autophagy has a protective function in neurons and its disruption results in neurodegenerative disorders. Neuronal damage is well-documented in the brains of HIV-infected individuals, and evidence of inflammation, oxidative stress, damage to synaptic and dendritic structures, and neuronal loss are present in the brains of those with HIV-associated dementia. We investigated the role of autophagy in microglia-induced neurotoxicity in primary rodent neurons, primate and human models. We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival. Quantitative analysis of autophagy vacuole numbers in rat primary neurons revealed a striking loss from the processes. Assessment of multiple biochemical markers of autophagic activity confirmed the inhibition of autophagy in neurons. Importantly, autophagy could be induced in neurons through rapamycin treatment, and such treatment conferred significant protection to neurons. Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-alpha and glutamate, had similar effects on reducing autophagy in neurons. The mRNA level of p62 was increased in the brain in SIV encephalitis and as well as in brains from individuals with HIV dementia, and abnormal neuronal p62 dot structures immunoreactivity was present and had a similar pattern with abnormal ubiquitinylated proteins. Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.

Multiple sclerosis (MS) is an inflammatory central nervous system (CNS) disorder characterized by T cell-mediated demyelination. In MS, prolonged T cell survival and increased T cell proliferation have been linked to disease relapse and progression. Recently, the autophagy-related gene 5 (Atg5) has been shown to modulate T cell survival. In this study, we examined the expression of Atg5 using both a mouse model of autoimmune demyelination as well as blood and brain tissues from MS cases. Quantitative real-time PCR analysis of RNA isolated from blood samples of experimental autoimmune encephalomyelitis (EAE) mice revealed a strong correlation between Atg5 expression and clinical disability.Analysis of protein extracted from these cells confirmed both upregulation and post-translational modification of Atg5, the latter of which was positively correlated with EAE severity. Analysis of RNA extracted from T cells isolated by negative selection indicated that Atg5 expression was significantly elevated in individuals with active relapsing-remitting MS compared to non-diseased controls. Brain tissue sections from relapsing-remitting MS cases examined by immunofluorescent histochemistry suggested that encephalitogenic T cells are a source of Atg5 expression in MS brain samples. Together these data suggest that increased T cell expression of Atg5 may contribute to inflammatory demyelination in MS.

Autophagy is emerging as a central regulator of cellular health and disease and, in the central nervous system (CNS), this homeostatic process appears to influence synaptic growth and plasticity. Herein, we review the evidence that dysregulation of autophagy may contribute to several neurodegenerative diseases of the CNS. Up-regulation of autophagy may prevent, delay or ameliorate at least some of these disorders, and - based on recent findings from our laboratory - we speculate that this goal may be achieved using a safe, simple and inexpensive approach.

Autophagy protects against many infections by inducing the lysosomal-mediated degradation of invading pathogens. However, previous in vitro studies suggest that some enteroviruses not only evade these protective effects but also exploit autophagy to facilitate their replication. We generated Atg5(f/f)/Cre(+) mice, in which the essential autophagy gene Atg5 is specifically deleted in pancreatic acinar cells, and show that coxsackievirus B3 (CVB3) requires autophagy for optimal infection and pathogenesis. Compared to Cre(-) littermates, Atg5(f/f)/Cre(+) mice had an ∼2,000-fold lower CVB3 titer in the pancreas, and pancreatic pathology was greatly diminished. Both in vivo and in vitro, Atg5(f/f)/Cre(+) acinar cells had reduced intracellular viral RNA and proteins. Furthermore, intracellular structural elements induced upon CVB3 infection, such as compound membrane vesicles and highly geometric paracrystalline arrays, which may represent viral replication platforms, were infrequently observed in infected Atg5(f/f)/Cre(+) cells. Thus, CVB3-induced subversion of autophagy not only benefits the virus but also exacerbates pancreatic pathology.

Summary Autophagy signaling pathway is involved in cellular homeostasis, developmental processes, cellular stress responses, and immune pathways. The aim of this review is to summarize the relationship between autophagy and viruses. It is not possible to be fully comprehensive, or to provide a complete “overview of all viruses”. In this review, we will focus on the interaction of autophagy and viruses and survey how human viruses exploit multiple steps in the autophagy pathway to help viral propagation and escape immune response. We discuss the role that macroautophagy plays in cells infected with hepatitis C virus, hepatitis B virus, rotavirus gastroenteritis, immune cells infected with human immunodeficiency virus, and viral respiratory tract infections both influenza virus and coronavirus.

Many recent studies indicate that dysregulation of autophagy is a common feature of many neurodegenerative diseases. The HIV-1-associated neurological disorder is an acquired cognitive and motor disease that includes a severe neurodegenerative dementia. We find that the neurodegeneration seen in the brain in HIV-1 infection is associated with an inhibition of neuronal autophagy, leading to neuronal demise. Neurons treated with supernatants from SIV-infected microglia develop a decrease in autophagy-inducing proteins, a decrease in neuronal autophagy vesicles, and an increase in sequestosome-1/p62. Examination of brains from HIV-infected individuals and SIV-infected monkeys reveals signs of autophagy dysregulation, associated, respectively, with dementia and encephalitis. Excitotoxic and inflammatory factors could inhibit neuronal autophagy, and stimulation of autophagy with rapamycin prevents such effects. Here we amplify on these findings, and propose that in the setting of HIV-infection, the decreased neuronal autophagy sensitizes cells to pro-apoptotic and other damaging mechanisms, leading to neuronal dysfunction and death. Hence, new therapeutic approaches aimed at boosting neuronal autophagy are conceivable to treat those suffering from the neurological complications of HIV.

RNA viruses modify intracellular membranes to produce replication scaffolds. In pancreatic cells, coxsackievirus B3 (CVB3) hijacks membranes from the autophagy pathway, and in vivo disruption of acinar cell autophagy dramatically delays CVB3 replication. This is reversed by expression of GFP-LC3, indicating that CVB3 may acquire membranes from an alternative, autophagy-independent, source(s). Herein, using 3 recombinant CVB3s (rCVB3s) encoding different proteins (proLC3, proLC3G120A, or ATG4BC74A), we show that CVB3 is, indeed, flexible in its utilization of cellular membranes. When compared with a control rCVB3, all 3 viruses replicated to high titers in vivo, and caused severe pancreatitis. Most importantly, each virus appeared to subvert membranes in a unique manner. The proLC3 virus produced a large quantity of LC3-I which binds to phosphatidylethanolamine (PE), affording access to the autophagy pathway. The proLC3G120A protein cannot attach to PE, and instead binds to the ER-resident protein SEL1L, potentially providing an autophagy-independent source of membranes. Finally, the ATG4BC74A protein sequestered host cell LC3-I, causing accumulation of immature phagophores, and massive membrane rearrangement. Taken together, our data indicate that some RNA viruses can exploit a variety of different intracellular membranes, potentially maximizing their replication in each of the diverse cell types that they infect in vivo.

Previous work has demonstrated that the surface glycoprotein (gp120) of human immunodeficiency virus-1 (HIV-1) can induce damage and apoptosis of neurons both in vitro and in vivo . In this report, we provide evidence that double-stranded RNA-activated protein kinase (PKR), a stress kinase, is involved in HIV/gp120-associated neurodegeneration. In cultures of mixed cortical cells, HIV/gp120 increased the protein level of PKR. Additionally, PKR was phosphorylated in neurons but not glia after exposure to gp120. The use of two independent pharmacological inhibitors of PKR activity abrogated neuronal cell death induced by gp120. Cortical neurons from PKR knock-out mice were significantly protected from neurotoxicity induced by gp120, further validating the pivotal proapoptotic function of PKR. gp120-induced phosphorylated PKR localized prominently to neuronal nuclei; PKR inhibition or the NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5 H -dibenzo [a,d] cyclohepten-5,10-imine maleate] abrogated this effect. PKR inactivation also inhibited gp120-induced caspase-3 activation, consistent with its neuroprotective effect. Finally, brain tissue from individuals diagnosed with HIV-associated dementia (HAD), but not HIV infection alone, contained the activated form of PKR, which localized predominantly to neuronal nuclei. Together, these results identify PKR as a critical mediator of gp120 neurotoxicity, suggesting that activation of PKR contributes to the neuronal injury and cell death observed in HAD.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme, has been implicated in the pathogenesis of various neurological disorders. IDO expression is induced by IFN-gamma and leads to neurotoxicity by generating quinolinic acid. Additionally, it inhibits the immune response through both tryptophan depletion and generating other tryptophan catabolites. IL-4 and IL-13 have been shown to control IDO expression by antagonizing the effects of IFN-gamma in different cell types. Here, we investigated the effects of these cytokines on IDO expression in microglia. Interestingly, we observed that both IL-4 and IL-13 greatly enhanced IFN-gamma-induced IDO expression. However, tryptophanyl-tRNA synthetase (WRS), which is coinduced with IDO by IFN-gamma, is downregulated by IL-4 and IL-13. The effect of IL-4 and IL-13 was independent of STAT-6. Modulation of IDO but not WRS was eliminated by inhibition of protein phosphatase 2A (PP2A) activity. The phosphatidylinositol 3-kinase (PI3K) pathway further differentiated the regulation of these two enzymes, as inhibiting the PI3K pathway eliminated IFN-gamma induction of IDO, whereas such inhibition greatly enhanced WRS expression. These findings show discordance between modulations of expression of two distinct enzymes utilizing tryptophan as a common substrate, and raise the possibility of their involvement in regulating immune responses in various neurological disorders.

Coxsackieviruses are important human pathogens, and their interactions with the innate and adaptive immune systems are of particular interest. Many viruses evade some aspects of the innate response, but coxsackieviruses go a step further by actively inducing, and then exploiting, some features of the host cell response. Furthermore, while most viruses encode proteins that hinder the effector functions of adaptive immunity, coxsackieviruses and their cousins demonstrate a unique capacity to almost completely evade the attention of naive CD8(+) T cells. In this artcle, we discuss the above phenomena, describe the current status of research in the field, and present several testable hypotheses regarding possible links between virus infection, innate immune sensing and disease.

Acute coxsackievirus B3 (CVB3) infection is one of the most prevalent causes of acute myocarditis, a disease that frequently is identified only after the sudden death of apparently healthy individuals. CVB3 infects cardiomyocytes, but the infection is highly focal, even in the absence of a strong adaptive immune response, suggesting that virus spread within the heart may be tightly constrained by the innate immune system. Type I interferons (T1IFNs) are an obvious candidate, and T1IFN receptor (T1IFNR) knockout mice are highly susceptible to CVB3 infection, succumbing within a few days of challenge. Here, we investigated the role of T1IFNs in the heart using a mouse model in which the T1IFNR gene can be ablated in vivo, specifically in cardiomyocytes. We found that T1IFN signaling into cardiomyocytes contributed substantially to the suppression of viral replication and infectious virus yield in the heart; in the absence of such signaling, virus titers were markedly elevated by day 3 postinfection (p.i.) and remained high at day 12 p.i., a time point at which virus was absent from genetically intact littermates, suggesting that the T1IFN-unresponsive cardiomyocytes may act as a safe haven for the virus. Nevertheless, in these mice the myocardial infection remained highly focal, despite the cardiomyocytes' inability to respond to T1IFN, indicating that other factors, as yet unidentified, are sufficient to prevent the more widespread dissemination of the infection throughout the heart. The absence of T1IFN signaling into cardiomyocytes also was accompanied by a profound acceleration and exacerbation of myocarditis and by a significant increase in mortality.Acute coxsackievirus B3 (CVB3) infection is one of the most common causes of acute myocarditis, a serious and sometimes fatal disease. To optimize treatment, it is vital that we identify the immune factors that limit virus spread in the heart and other organs. Type I interferons play a key role in controlling many virus infections, but it has been suggested that they may not directly impact CVB3 infection within the heart. Here, using a novel line of transgenic mice, we show that these cytokines signal directly into cardiomyocytes, limiting viral replication, myocarditis, and death.

Human immunodeficiency virus (HIV)-associated dementia (HAD) is a syndrome occurring in HIV-infected patients with advanced disease that likely develops as a result of macrophage and microglial activation as well as other immune events triggered by virus in the central nervous system. The most relevant experimental model of HAD, rhesus macaques exhibiting simian immunodeficiency virus (SIV) encephalitis (SIVE), closely reproduces the human disease and has been successfully used to advance our understanding of mechanisms underlying HAD. In this study we integrate gene expression data from uninfected and SIV-infected hippocampus with a human protein interaction network and discover modules of genes whose expression patterns distinguish these two states, to facilitate identification of neuronal genes that may contribute to SIVE/HIV cognitive deficits. Using this approach we identify several downregulated candidate genes and select one, EGR1, a key molecule in hippocampus-related learning and memory, for further study. We show that EGR1 is downregulated in SIV-infected hippocampus and that it can be downregulated in differentiated human neuroblastoma cells by treatment with CCL8, a product of activated microglia. Integration of expression data with protein interaction data to discover discriminatory modules of interacting proteins can be usefully used to prioritize differentially expressed genes for further study. Investigation of EGR1, selected in this manner, indicates that its downregulation in SIVE may occur as a consequence of the host response to infection, leading to deficits in cognition.

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate and zinc, as well as H(2)O(2) formation during the reperfusion period, strongly depresses protein synthesis. We have previously demonstrated that the glutamate-induced increase in cytosolic Ca(2+) is likely responsible for blockade of the elongation step of protein synthesis, whereas Zn(2+) preferentially inhibits the initiation step. In this study, we provide evidence indicating that H(2)O(2) and thapsigargin mobilized a common intracellular Ca(2+) pool. H(2)O(2) treatment stimulated a slow increase in intracellular Ca(2+), and precluded the effect of thapsigargin on Ca(2+) mobilization. H(2)O(2) stimulated the phosphorylation of both eIF-2alpha and eEF-2, in a time- and dose-dependent manner, suggesting that both the blockade of the elongation and of the initiation step are responsible for the H(2)O(2)-induced inhibition of protein synthesis. However, kinetic data indicated that, at least during the first 15 min of H(2)O(2) treatment, the inhibition of protein synthesis resulted mainly from the phosphorylation of eEF-2. In conclusion, H(2)O(2) inhibits protein translation in cortical neurons by a process that involves the phosphorylation of both eIF-2alpha and eEF-2 and the relative contribution of these two events depends on the duration of H(2)O(2) treatment.

Autophagy plays a protective role during many viral and bacterial infections. Predictably, evolution has led to several viruses developing mechanisms by which to evade the inhibitory effects of the pathway. However, one family of viruses, the picornaviruses, has gone one step further, by actively exploiting autophagy. Using mice in which Atg5 has been conditionally deleted in pancreatic acinar cells, we have studied the outcome of infection by coxsackievirus B3 (CVB3), a member of the enterovirus genus and picornavirus family. Two key findings emerged: disruption of autophagy (1) dramatically compromised virus replication in vivo, and (2) significantly limited pancreatic disease.

Viral myocarditis is a serious disease, commonly caused by type B coxsackieviruses (CVB). Here we show that innate immune protection against CVB3 myocarditis requires the IFIT (IFN-induced with tetratricopeptide) locus, which acts in a biphasic manner. Using IFIT locus knockout (IFITKO) cardiomyocytes we show that, in the absence of the IFIT locus, viral replication is dramatically increased, indicating that constitutive IFIT expression suppresses CVB replication in this cell type. IFNβ pre-treatment strongly suppresses CVB3 replication in wild type (wt) cardiomyocytes, but not in IFITKO cardiomyocytes, indicating that other interferon-stimulated genes (ISGs) cannot compensate for the loss of IFITs in this cell type. Thus, in isolated wt cardiomyocytes, the anti-CVB3 activity of IFITs is biphasic, being required for protection both before and after T1IFN signaling. These in vitro findings are replicated in vivo. Using novel IFITKO mice we demonstrate accelerated CVB3 replication in pancreas, liver and heart in the hours following infection. This early increase in virus load in IFITKO animals accelerates the induction of other ISGs in several tissues, enhancing virus clearance from some tissues, indicating that–in contrast to cardiomyocytes–other ISGs can offset the loss of IFITs from those cell types. In contrast, CVB3 persists in IFITKO hearts, and myocarditis occurs. Thus, cardiomyocytes have a specific, biphasic, and near-absolute requirement for IFITs to control CVB infection.

In the central nervous system, Zn2+ is concentrated in the cerebral cortex and hippocampus and has been found to be toxic to neurons. In this study, we show that exposure of cultured cortical neurons from mouse to increasing concentrations of Zn2+ (10–300 μm) induces a progressive decrease in global protein synthesis. The potency of Zn2+ was increased by about 2 orders of magnitude in the presence of Na+-pyrithione, a Zn2+ ionophore. The basal rate of protein synthesis was restored 3 h after Zn2+ removal. Zn2+induced a sustained increase in phosphorylation of the α subunit of the translation eukaryotic initiation factor-2 (eIF-2α), whereas it triggered a transient increase in phosphorylation of eukaryotic elongation factor-2 (eEF-2). Protein synthesis was still depressed 60 min after the onset of Zn2+ exposure while the state of eEF-2 phosphorylation had already returned to its basal level. Moreover, Zn2+ was less effective than glutamate to increase eEF-2 phosphorylation, whereas it induced a more profound inhibition of protein synthesis. These results suggest that Zn2+-induced inhibition of protein synthesis mainly correlates with the increase in eIF-2α phosphorylation. Supporting further that Zn2+ acts at the initiation step of protein synthesis, it strongly decreased the amount of polyribosomes. In the central nervous system, Zn2+ is concentrated in the cerebral cortex and hippocampus and has been found to be toxic to neurons. In this study, we show that exposure of cultured cortical neurons from mouse to increasing concentrations of Zn2+ (10–300 μm) induces a progressive decrease in global protein synthesis. The potency of Zn2+ was increased by about 2 orders of magnitude in the presence of Na+-pyrithione, a Zn2+ ionophore. The basal rate of protein synthesis was restored 3 h after Zn2+ removal. Zn2+induced a sustained increase in phosphorylation of the α subunit of the translation eukaryotic initiation factor-2 (eIF-2α), whereas it triggered a transient increase in phosphorylation of eukaryotic elongation factor-2 (eEF-2). Protein synthesis was still depressed 60 min after the onset of Zn2+ exposure while the state of eEF-2 phosphorylation had already returned to its basal level. Moreover, Zn2+ was less effective than glutamate to increase eEF-2 phosphorylation, whereas it induced a more profound inhibition of protein synthesis. These results suggest that Zn2+-induced inhibition of protein synthesis mainly correlates with the increase in eIF-2α phosphorylation. Supporting further that Zn2+ acts at the initiation step of protein synthesis, it strongly decreased the amount of polyribosomes. α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid N-methyl-d-aspartate eukaryotic elongation factor-2 α subunit of the eukaryotic initiation factor-2 N-(6-methoxy-8-quinolyl)-p-toluene sulfonamide N,N,N′,N′-tetrakis(2-pyridyl-methyl)ethylene diamine (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate 6,7-dinitroquinoxaline-2,3-dione RNA-dependent protein kinase PKR-like endoplasmic reticulum kinase long term potentiation 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid The transition metal Zn2+ is widely but heterogeneously distributed in the brain. It is mainly detected in glutamatergic neurons of the neocortex and in nerve terminals of hippocampal mossy fibers (1Frederickson C.J. Moncrieff D.W. Biol. Signals. 1994; 3: 127-139Crossref PubMed Scopus (196) Google Scholar). In these latter nerve terminals, Zn2+ appears to be contained in synaptic vesicles and is released with glutamate during neuronal activity (2Assaf S.Y. Chung S.H. Nature. 1984; 308: 734-736Crossref PubMed Scopus (1012) Google Scholar, 3Howell G.A. Welch M.G. Frederickson C.J. Nature. 1984; 308: 736-738Crossref PubMed Scopus (696) Google Scholar). Several studies suggest that Zn2+ may modulate excitatory neurotransmission. Zn2+ inhibits glutamate uptake into glial cells (4Spiridon M. Kamm D. Billups B. Mobbs P. Attwell D. J. Physiol. 1998; 506: 363-376Crossref PubMed Scopus (83) Google Scholar) and synaptosomes (5Gabrielsson B. Robson T. Norris D. Chung S.H. Brain Res. 1986; 384: 218-223Crossref PubMed Scopus (30) Google Scholar) and facilitates α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid (AMPA)1 receptor-mediated neuroexcitation (6Rassendren F.A. Lory P. Pin J.P. Nargeot J. Neuron. 1990; 4: 733-740Abstract Full Text PDF PubMed Scopus (149) Google Scholar). Alternatively, Zn2+ has been shown to inhibit N-methyl-d-aspartate (NMDA) receptor function through both voltage-dependent and voltage-independent mechanisms (7Christine C.W. Choi D.W. J. Neurosci. 1990; 10: 108-116Crossref PubMed Google Scholar, 8Ascher P. Nat. Neurosci. 1998; 1: 173-175Crossref PubMed Scopus (8) Google Scholar). Beside its modulatory effects on glutamatergic transmission, Zn2+ has been found to contribute to neuronal loss induced by transient cerebral ischemia (9Koh J.-Y. Suh S.W. Gwag B.J. He Y.Y. Hsu C.Y. Choi D.W. Science. 1996; 272: 1013-1016Crossref PubMed Scopus (921) Google Scholar). The precise mechanism responsible for the neurotoxic effect of Zn2+ is unknown. However, NMDA receptor antagonists appear to exert a protecting effect against Zn2+-induced neurotoxicity (10Koh J.-Y Choi D.W. Neuroscience. 1994; 60: 1049-1057Crossref PubMed Scopus (192) Google Scholar). At the intracellular level, Zn2+ can interact with a large variety of factors including metallothioneins, reduced glutathione and ion transporter enzymes such as Na+/K+-ATPase and Ca2+-ATPase (for review, see Ref. 11Vallee B.L. Falchuk K.H. Physiol. Rev. 1993; 73: 79-117Crossref PubMed Google Scholar). In neurons, Zn2+ treatment and NMDA receptor stimulation lead to an inhibition of cell respiration and thus of ATP synthesis. However, Zn2+ inhibits the cell respiratory chain by blocking the initial step of respiration, i.e. the electron transfer between ubiquinone and cytochrome b (complex III) (12Cuajungco M.P. Lees G.J. Neurobiol. Dis. 1997; 4: 137-169Crossref PubMed Scopus (514) Google Scholar), whereas glutamate induces a loss of the mitochondrial potential by opening the transition pore (13White R.J. Reynolds I.J. J. Neurosci. 1996; 15: 5688-5697Crossref Google Scholar, 14Schinder A.F. Olson E.C. Spitzer N.C. Montal M. J. Neurosci. 1996; 16: 6125-6133Crossref PubMed Google Scholar). We have previously demonstrated that glutamate, by stimulating NMDA- and AMPA-gated channels, depresses global protein synthesis in cultured cortical neurons from the mouse (15Marin P. Nastiuk K.L. Daniel N. Girault J.A. Czernik A.J. Glowinski J. Nairn A.C. Prémont J. J. Neurosci. 1997; 17: 3445-3454Crossref PubMed Google Scholar). This effect appears to result from the phosphorylation of eukaryotic elongation factor-2 (eEF-2) by eEF-2 kinase, a Ca2+-calmodulin-dependent enzyme (15Marin P. Nastiuk K.L. Daniel N. Girault J.A. Czernik A.J. Glowinski J. Nairn A.C. Prémont J. J. Neurosci. 1997; 17: 3445-3454Crossref PubMed Google Scholar). It has also been reported that Zn2+ inhibits protein synthesis in reticulocyte lysate by a distinct mechanism, which involves the phosphorylation of the α subunit of the eukaryotic initiation factor-2 (eIF-2α) (16Hurst R. Schatz J.R. Matts R.L. J. Biol. Chem. 1987; 262: 15939-15945Abstract Full Text PDF PubMed Google Scholar). The aim of the present study was to determine whether Zn2+ depresses protein synthesis in living cortical neurons and to investigate the mechanism involved in this process. Primary neuronal cultures were prepared as described previously (17Weiss S. Pin J.P. Sebben M. Kemp D.E. Sladeczek F. Gabrion J. Bockaert J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2238-2242Crossref PubMed Scopus (170) Google Scholar). Briefly, cortices were removed from 15-day-old Swiss mouse embryos (Iffa Credo, Lyon, France) and cells were seeded on 6- or 12-well culture dishes (3 × 106 and 1 × 106 cells/well containing 3 and 1 ml of culture medium, respectively), coated successively with poly-l-ornithine (15 μg/ml, Mr = 40,000, Sigma) and culture medium containing 10% fetal calf serum (Dutcher, Brumath, France). The culture medium included a 1:1 mixture of Dulbecco's modified Eagle's medium and F-12 nutrient (Life Technologies, Inc., Paris, France), supplemented with glucose (33 mm) glutamine (2 mm), NaHCO3 (13 mm), HEPES buffer (5 mm, pH 7.4), penicillin-streptomycin (5 IU/ml and 5 μg/ml, respectively), and a mixture of salt and hormones containing insulin (25 μg/ml), transferrin (100 μg/ml), progesterone (20 nm), putrescine (60 μm), and Na2SeO3 (30 nm). Cells were maintained for 6 days at 37 °C in a humidified atmosphere containing 8% CO2 without medium change. In these conditions, the cultures were shown to be highly enriched in neurons by immunocytochemistry using an anti-microtubule-associated protein 2 monoclonal antibody (IgG1, Biomakor, Israel). Less than 7% of the cells exhibited immunoreactivity with a rabbit antibody raised against glial fibrillary acid protein (Dakopatts, Glostrup, Denmark) (data not shown). Neurons grown in 12-well culture dishes were washed twice in 1 ml of HEPES buffer (in mm: HEPES, 20; glucose, 5.5; NaCl, 120; KCl, 5.5; MgCl2, 0.9; CaCl2, 1.1; pH 7.4) and then incubated for 30 min in this medium in the presence of drugs and 50 μm either methionine or leucine. [35S]methionine (1000 Ci/mmol, Amersham Pharmacia Biotech) or [3H]leucine (159 Ci/mmol, Amersham Pharmacia Biotech) were added (4 μCi/ml each) during the last 10 min of the incubation period. The labeling was stopped by 2 washes in 1 ml of ice-cold phosphate buffered saline and addition of 1 ml of ice-cold trichloroacetic acid (10%, w/v). Cells were scraped, and suspensions were centrifuged for 10 min at 10,000 × g. Amino acid uptake into neurons and incorporation into proteins were estimated by counting the radioactivity in the supernatant and the pellet, respectively. Results are expressed as the ratio between the radioactive amino acid incorporated into proteins (trichloroacetic acid-precipitable fraction) and the radioactive amino acid taken up into the cells (supernatant). Intracellular Zn2+ was detected in neurons grown on glass slides using the Zn2+-selective and membrane-permeant fluorescent dyeN-(6-methoxy-8-quinolyl)-p-toluene sulfonamide (TSQ). Glass slides were placed in a superfusion chamber where cells were superfused with HEPES buffer. Neurons were exposed to drugs including TSQ (0.001%, wt/v, prepared from a stock solution of 0.5% in dimethyl sulfoxide) using a multichannel superfusion device. The superfusion chamber was placed on the stage of a Nikon Diaphot inverted microscope equipped with a 75-watt xenon light and a 40× epifluorescence oil immersion objective. Light was filtered at 360 nm with a 10-nm-wide interferential filter and emission light was passed through a 380-nm dichroic long pass filter (barrier 420 nm). Images were acquired with an intensified CCD camera and digitized using an Argus 50 interface (16 video frames per digitized image, allowing the recording of 1 image/s). The camera and the digitizing system were from Hamamatsu (Japan). The camera dark noise was subtracted from the recorded crude image at the beginning of each experiment. Cortical neurons grown in six-well culture dishes were labeled for 3.5 h with [32P]orthophosphate (200 μCi/ml) in 1 ml of HEPES buffer. Drugs were then added to cells for the indicated times. Neurons were lyzed in 200 μl of immunoprecipitation buffer containing 100 mm NaCl, 50 mm Tris-HCl, pH 7.6, 5 mm EDTA, 50 mm NaF, 50 mmβ-glycerophosphate, 1 mm benzamidine, 0.5 mmNa+-vanadate, 1% Triton X-100, and a protease inhibitor mixture (Roche Molecular Biochemicals), and centrifuged for 10 min at 10,000 × g. Supernatant proteins (100 μg) were immunoprecipitated overnight with an antibody recognizing specifically eIF-2α (10 μg of purified immunoglobulin per sample) and 30 μl of protein A-Sepharose beads. The serum recognizing eIF-2α was obtained by immunization of rabbits with a synthetic peptide derived from the sequence of the protein (LSKRRVSPEEAIKC) and purified on an affinity column. The purified antibody recognized an unique band around 36 kDa in whole homogenates prepared from cultured cortical neurons (data not shown). Immunocomplexes were washed three times in immunoprecipitation buffer, boiled in SDS loading buffer (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar), and resolved on 10% polyacrylamide gels. Incorporation of 32P into eIF-2α was detected by autoradiography and quantified by PhosphorImager. The amount of immunoprecipitated eIF-2α was estimated in an aliquot of each samples by Western blotting using a monoclonal antibody directed against eIF-2α (20Price N.T. Proud C.G. Biochim. Biophys. Acta. 1990; 1054: 83-88Crossref PubMed Scopus (18) Google Scholar). eIF-2α phosphorylation state was also analyzed by sequential immunoblotting, first with an antibody recognizing specifically the phosphorylated form of eIF-2α (1/250 dilution, Research Genetics Inc., Huntsville, AL) and then with the polyclonal antibody recognizing total eIF-2α (1/2,000 dilution). Neurons, grown in six-well culture dishes, were exposed to drugs in HEPES buffer for the indicated times. Incubations were stopped by replacing the medium by 0.3 ml of boiling SDS (1%, w/v), in order to prevent protein dephosphorylation by phosphatases. Protein concentration was determined with a bicinchoninic acid method (19Smith P.K. Krohn R.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18349) Google Scholar), using bovine serum albumin as standard. Samples containing 50 μg of protein were resolved on 8% polyacrylamide gels and transferred to nitrocellulose. Antibody-antigen complexes were detected with an enhanced chemiluminescence method (Renaissance kit from NEN Life Science Products) using a horseradish peroxidase-coupled donkey anti-rabbit secondary antibody (Amersham Pharmacia Biotech). The phosphorylation of eEF-2 in living cortical neurons was analyzed by sequential immunoblotting first with an antibody that specifically recognized eEF-2 phosphorylated on Thr56 (1/1,000 dilution) and then with an antibody recognizing eEF-2 independently of its phosphorylation state (1/1,000 dilution) (15Marin P. Nastiuk K.L. Daniel N. Girault J.A. Czernik A.J. Glowinski J. Nairn A.C. Prémont J. J. Neurosci. 1997; 17: 3445-3454Crossref PubMed Google Scholar), as described above. The amount of RNA associated with the polyribosomal fraction was estimated as described previously (21Weiler I.J. Greenough W.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7168-7171Crossref PubMed Scopus (209) Google Scholar, 22Weiler I.J. Childers W.S. Greenough W.T. J. Neurochem. 1996; 66: 197-202Crossref PubMed Scopus (14) Google Scholar). Cortical neurons grown for 6 days in 60-mm culture dishes were exposed for 30 min to 100 μm cycloheximide in the absence or presence of 100 μm ZnCl2. Cells were lyzed in a lysing buffer containing 0.25% CHAPS, 125 mm NaCl, 100 mm sucrose, 2 mm potassium acetate, 50 mm HEPES (pH 7.5), 5 mm MgCl2, 1 mm sodium vanadate, 2 mm dithiothreitol, and 100 μm cycloheximide, and immediately spun for 30 min in a microcentrifuge. The supernatant was layered over 1 msucrose containing 10 mm Tris (pH 7.6), 2 mmdithiothreitol, 2 mm potassium acetate, and 5 mm MgCl2, and centrifuged for 10 min at 400,000 × g in a Beckman TL-100 ultracentrifuge. RNA were extracted from the resultant polyribosomal pellet as described previously (23Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar) and resolved on 1% agarose gels. The amount of 28 and 18 S ribosomal RNA was estimated by scanning of ethidium bromide fluorescence. The exposure of cultured cortical neurons to Zn2+ for 30 min resulted in a marked decrease in [3H]leucine (Fig. 1) or [35S]methionine (data not shown) incorporation into proteins. This inhibition of neuronal protein synthesis was concentration-dependent (IC50 = 51 ± 7 μm, mean ± S.E. of values obtained in five independent experiments performed in triplicate), and the potency of Zn2+ was increased (1–10 μm range, Fig. 1) in the presence of 20 μm Na+-pyrithione, a Zn2+ ionophore. As detected using the Zn2+-sensitive fluorescent dye TSQ, the Na+-pyrithione treatment strongly increased the staining of neurons exposed to 3 μm Zn2+ (Fig.2). It should be noted that, at this low concentration (3 μm), Zn2+ inhibited by 50% the protein synthesis in the presence of Na+-pyrithione, whereas it was ineffective in its absence. None of these treatments (Zn2+ with or without Na+-pyrithione) significantly altered the uptake of radioactive amino acids into neurons (data not shown).Figure 2Enhancement of Zn2+ entry in cortical neurons by Na+-pyrithione. Cortical neurons grown on glass slides were first superfused with TSQ to assess basal level of Zn2+. They were then exposed for 1 min to 3 μm ZnCl2 in either the absence (a) or the presence (b) of 20 μmNa+-pyrithione and thereafter to TSQ until the fluorescence reaches its maximal level (about 1–2 min). A representative field of three independent experiments performed on different sets of cultured neurons, photographed by fluorescence videomicroscopy, is illustrated. Scale bar = 30 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Zn2+ influx into neurons has been shown to occur through AMPA- or NMDA-gated channels and L-type voltage-dependent Ca2+ channels (24Sensi S.L. Canzoniero L.M.T., Yu, S.P. Ying H.S. Koh J.-Y. Kerchner G.A. Choi D.W. J. Neurosci. 1997; 17: 9554-9564Crossref PubMed Google Scholar). However, neither nifedipine, an antagonist of L-type voltage-gated Ca2+ channels, nor (+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]-cyclohepten-5,10-imine hydrogen maleate (MK-801) or 6,7-dinitroquinoxaline-2,3-dione (DNQX), antagonists of NMDA and AMPA receptors, respectively, suppressed the inhibition of protein synthesis induced by 100 μm Zn2+ (Fig.3). Additional experiments were performed to determine whether these treatments modify the Zn2+-induced TSQ fluorescence. Since TSQ binds Zn2+ in a saturable process, variations of Zn2+bound to TSQ must be investigated in the presence of Zn2+concentrations that are largely lower than those leading to the saturation of the dye (used at 30 μm). Thus, in order to warrant absence of saturation of the dye, we have investigated the mechanisms of Zn2+ entry in neurons exposed to 3 μm Zn2+ (see Fig. 2). In this experimental condition, the co-application of DNQX and nifedipine with Zn2+ did not decrease TSQ fluorescence (Fig.4). Only MK-801 decreased by 38 ± 7% (n = 33 cells tested) the fluorescence signal of the dye.Figure 4Role of ionotropic glutamatergic receptors and voltage-gated Ca2+ channels in Zn2+ influx in cortical neurons. In a first step, cortical neurons were superfused with 3 μmZnCl2 in the absence or presence of either 10 μm nifedipine (Nife), 100 μmDNQX, or 2 μm MK-801, and then with TSQ (until the fluorescence reaches a maximal level). In a second step, they were exposed to 3 μm ZnCl2 alone and then to TSQ. TSQ fluorescence was quantified in cell bodies. Results were expressed as a ratio of fluorescence intensities obtained successively in the same neurons with or without the indicated antagonist. They are the mean ± S.E. of values calculated in 30–35 neurons originating from two sets of cultured neurons. *, p < 0.05 compared with neurons only exposed to 3 μmZnCl2 (analysis of variance followed by Dunnett's test).View Large Image Figure ViewerDownload Hi-res image Download (PPT) A progressive decline of protein synthesis was observed when cortical neurons were continuously treated with 100 μm Zn2+, the maximal inhibition being reached after a 50-min exposure (Fig.5). When neurons were exposed to 100 μm Zn2+ for 30 min only, the marked decrease of protein synthesis already measured at the end of this treatment was followed by an almost complete recovery to control levels 3 h after the removal of Zn2+ (Fig. 5). This recovery was accelerated (less than 1 h) by adding the Zn2+chelator, N,N,N′,N′-tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN, 10 μm), immediately after the removal of Zn2+(Fig. 5). TSQ fluorescence following Zn2+ removal declined very slowly (92 ± 7% of initial TSQ fluorescence was still detected 1 h after Zn2+ removal). Addition of TPEN just after Zn2+ removal lead to the almost complete disappearance of TSQ fluorescence in less than 2 min (data not shown). We have previously demonstrated that Ca2+ influx in cortical neurons resulting from the activation of ionotropic glutamate receptors inhibits protein synthesis. This effect was correlated with the increase in the phosphorylation of eEF-2 by eEF-2 kinase, a Ca2+-calmodulin-dependent enzyme (15Marin P. Nastiuk K.L. Daniel N. Girault J.A. Czernik A.J. Glowinski J. Nairn A.C. Prémont J. J. Neurosci. 1997; 17: 3445-3454Crossref PubMed Google Scholar). This Ca2+-mediated process is likely not the main mechanism responsible for Zn2+-induced inhibition of protein synthesis. Indeed, after a 1-h exposure of cortical neurons to Zn2+, eEF-2 was not phosphorylated, whereas protein synthesis was still depressed (Figs. 5 and6). A significant increase in eEF-2 phosphorylation was detected after a 30-min exposure of neurons to 100 μm Zn2+ (Fig. 6) but this phosphorylation was of lower amplitude than that evoked by a maximally effective concentration of glutamate (100 μm, Fig. 6), which only depressed by 50% the rate of protein synthesis in cortical neurons (15Marin P. Nastiuk K.L. Daniel N. Girault J.A. Czernik A.J. Glowinski J. Nairn A.C. Prémont J. J. Neurosci. 1997; 17: 3445-3454Crossref PubMed Google Scholar). Moreover, the exposure of neurons to 100 μmZn2+ resulted in a strong decrease of RNA associated with the polyribosomal fraction (Fig. 7), indicating that the initiation but not the elongation step of protein synthesis is likely involved in Zn2+-induced inhibition of protein synthesis.Figure 7Effect of Zn2+ on the amount of polyribosome in cortical neurons. Cortical neurons were exposed to sham treatment or 100 μm ZnCl2 for 30 min. Polyribosomal RNA were isolated as described under “Experimental Procedures.” Top, revelation of polyribosomal RNA by ethidium bromide staining. Bottom, quantification of the fluorescence intensities of (28 S + 18 S) bands (expressed as percentage of basal). *, p < 0.01, Student's t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Exposure of cortical neurons to a Ca2+-free buffer or to thapsigargin, two treatments which are known to deplete intracellular Ca2+ stores, led to a marked decrease in protein synthesis (Figs. 3 and 8). It has been demonstrated in other cell types that these treatments increase the phosphorylation of eIF-2α (25Srivastava S.P. Davies M.V. Kaufman R.J. J. Biol. Chem. 1995; 270: 16619-16624Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 26Prostko C.R. Dholakia J.N. Brostrom M.A. Brostrom C.O. J. Biol. Chem. 1995; 270: 6211-6215Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Similarly, exposure of neurons to Ca2+-free buffer or to thapsigargin increased the incorporation of 32P into eIF-2α by 2.6-fold (data not shown) and 4.1-fold (Fig. 8, a and c), respectively. As shown in rabbit reticulocyte lysate, heavy metals including Zn2+ increase the phosphorylation level of eIF-2α, a process that could account for their ability to inhibit protein translation (16Hurst R. Schatz J.R. Matts R.L. J. Biol. Chem. 1987; 262: 15939-15945Abstract Full Text PDF PubMed Google Scholar). Similarly, the incorporation of32P into eIF-2α was enhanced when neurons were exposed to 100 μm Zn2+ for 30 min (Fig. 8, aand c). The enhanced phosphorylation of eIF-2α in neurons exposed to Zn2+ or thapsigargin was also observed in Western blotting experiments using an antibody that recognizes specifically the phosphorylated form of the protein (Fig.8 b). As observed for the Zn2+-induced inhibition of protein synthesis, eIF-2α phosphorylation still persisted after a 1-h exposure of cortical neurons to Zn2+ (3.2 ± 0.4-fold increase in 32P incorporation into eIF-2α, as compared with the basal level, n = 4). Arguing in favor of the role of eIF-2α phosphorylation in Zn2+-induced inhibition of protein synthesis, the magnitude of inhibition of protein synthesis induced by Zn2+ and thapsigargin was correlated with their ability to increase eIF-2α phosphorylation (Fig. 8, c and d). Moreover, the effects of both treatments were not additive (Fig. 8). Increased eIF-2α phosphorylation in neurons exposed to Zn2+ could result from either enhanced eIF-2α kinase activity or inhibition of phosphatase. Indeed, a 30-min exposure of neurons to 100 nmokadaic acid, a nonselective inhibitor of phosphatase 2A, induced an increase in eIF-2α phosphorylation and an inhibition of protein synthesis similar to those evoked by 100 μmZn2+ (data not shown). In this study, we show for the first time that the transient exposure of living cortical neurons to Zn2+ markedly decreases protein synthesis. This effect was observed with concentrations of Zn2+ (10–300 μm) that are in the range of those reached in the extracellular space under physiopathological conditions such as cerebral ischemia (2Assaf S.Y. Chung S.H. Nature. 1984; 308: 734-736Crossref PubMed Scopus (1012) Google Scholar). The Zn2+-induced inhibition of protein synthesis probably results from the interaction of this heavy metal with intracellular components, as it was enhanced in the presence of the Zn2+ionophore, Na+-pyrithione. This protein synthesis inhibition appears to be slowly reversible, as a recovery to the basal level of radioactive amino acid incorporation into proteins was observed 3 h after the transient exposure (30 min) of cortical neurons to Zn2+. The time course of this recovery process could reflect the slow dissociation rate of Zn2+ from its intracellular binding sites. Indeed, Zn2+ is known to be tightly bound to several intracellular components such as phospholipids (binding to phosphate head groups) (27Ludwig J.C. Chavapil M. Agents Actions. 1981; 9: 65-68Google Scholar), membrane-bound enzymes such as phospholipases (reaction with sulfhydryl groups leading to the formation of stable mercaptides) (28Warren L. Glick M. Nass M. J. Cell. Physiol. 1966; 68: 269-288Crossref Scopus (208) Google Scholar), or other thiol-containing molecules such as metallothioneins or reduced glutathione (11Vallee B.L. Falchuk K.H. Physiol. Rev. 1993; 73: 79-117Crossref PubMed Google Scholar). Supporting further this hypothesis, the Zn2+ chelator TPEN accelerated the recovery of protein synthesis to control levels. As already reported, Zn2+ enters neurons by several routes including NMDA and AMPA receptors and L-type voltage-gated Ca2+ channels (24Sensi S.L. Canzoniero L.M.T., Yu, S.P. Ying H.S. Koh J.-Y. Kerchner G.A. Choi D.W. J. Neurosci. 1997; 17: 9554-9564Crossref PubMed Google Scholar). However, the Zn2+-induced inhibition of protein synthesis in cortical neurons (observed in the absence of glutamatergic agonists and under non-depolarizing conditions) persisted in the presence of glutamate receptor antagonists or nifedipine, a blocker of L-type voltage-sensitive Ca2+channels. Similarly, DNQX and nifedipine did not alter TSQ fluorescence signal, but MK-801 partially decreased the fluorescence of the dye. However, for the aforementioned technical considerations, this set of experiments was done in the presence of a low concentration of Zn2+ as compared with those required to observe an inhibition of protein synthesis. Assuming that MK-801 blocks with the same efficiency Zn2+ entry in neurons exposed to 100 μm Zn2+, one can predict from the dose response for Zn2+ on protein synthesis that such a partial decrease of intracellular Zn2+ concentration does not significantly modify the rate of protein translation. Together, these results suggest that the process of Zn2+ influx into neurons that leads to the inhibition of protein synthesis is different from those previously described. We have previously reported that the global protein synthesis in neurons can be inhibited by a Ca2+-dependent process (15Marin P. Nastiuk K.L. Daniel N. Girault J.A. Czernik A.J. Glowinski J. Nairn A.C. Prémont J. J. Neurosci. 1997; 17: 3445-3454Crossref PubMed Google Scholar). The phosphorylation of the elongation factor eEF-2 by eEF-2 kinase, a Ca2+ -calmodulin-dependent kinase, appears to be involved in the glutamate-induced reduction of protein synthesis. This conclusion was based on the close correlation between the magnitude of protein synthesis inhibition and the level of eEF-2 phosphorylation (15Marin P. Nastiuk K.L. Daniel N. Girault J.A. Czernik A.J. Glowinski J. Nairn A.C. Prémont J. J. Neurosci. 1997; 17: 3445-3454Crossref PubMed Google Scholar). Two observations suggest that, although Zn2+ does transiently increase eEF-2 phosphorylation, this process is likely not the main mechanism responsible for Zn2+-induced inhibition of protein synthesis. First, a 100 μm Zn2+ treatment, which inhibited protein synthesis to a larger extent than that induced by a maximally effective concentration of glutamate (100 μm), led to an increase in eEF-2 phosphorylation significantly lower than that induced by this excitatory amino acid. Second, protein synthesis was still depressed 60 min after the onset of Zn2+ exposure, whereas the recovery to control levels of the phosphorylation state of eEF-2 was almost complete. The decrease in the amount of polyribosome in neurons exposed to Zn2+ suggests that the metal acts instead at the initiation step of protein synthesis, in which the phosphorylation of eIF-2α constitutes a key mechanism of regulation. Three kinases have been found to phosphorylate eIF-2α: the heme-regulated inhibitor of erythroid cells, the interferon-inducible RNA-dependent protein kinase (PKR), and the recently discovered PERK (for PKR-like endoplasmic reticulum kinase) (25Srivastava S.P. Davies M.V. Kaufman R.J. J. Biol. Chem. 1995; 270: 16619-16624Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 26Prostko C.R. Dholakia J.N. Brostrom M.A. Brostrom C.O. J. Biol. Chem. 1995; 270: 6211-6215Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 29Samuel C.E. J. Biol. Chem. 1993; 268: 7603-7606Abstract Full Text PDF PubMed Google Scholar, 30Proud C.G. Trends Biol. Sci. 1995; 20: 241-246Abstract Full Text PDF PubMed Scopus (200) Google Scholar, 31Harding H.P. Zang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2457) Google Scholar, 32Shi Y. Vattem K.M. Sood R. An J. Liang J. Stramm L. Wek R.C. Mol. Cell. Biol. 1998; 18: 7499-7509Crossref PubMed Google Scholar). It has been suggested that Ca2+ release from endoplasmic reticulum or the resulting depletion of intracellular Ca2+ stores induces the phosphorylation of eIF-2α (25Srivastava S.P. Davies M.V. Kaufman R.J. J. Biol. Chem. 1995; 270: 16619-16624Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 26Prostko C.R. Dholakia J.N. Brostrom M.A. Brostrom C.O. J. Biol. Chem. 1995; 270: 6211-6215Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 31Harding H.P. Zang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2457) Google Scholar). As initially proposed, PKR could be involved in this phosphorylation (25Srivastava S.P. Davies M.V. Kaufman R.J. J. Biol. Chem. 1995; 270: 16619-16624Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 26Prostko C.R. Dholakia J.N. Brostrom M.A. Brostrom C.O. J. Biol. Chem. 1995; 270: 6211-6215Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). However, according to two recent reports, there is strong evidence that the newly discovered eIF-2α kinase PERK is implicated in this cellular stress response (31Harding H.P. Zang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2457) Google Scholar, 32Shi Y. Vattem K.M. Sood R. An J. Liang J. Stramm L. Wek R.C. Mol. Cell. Biol. 1998; 18: 7499-7509Crossref PubMed Google Scholar). Thus, the activation of this kinase may be responsible for both the increase in eIF-2α phosphorylation and the inhibition of protein synthesis found in neurons exposed to either thapsigargin or a Ca2+-free medium. Zn2+ treatment also induced a prominent inhibition of protein synthesis associated with an increased phosphorylation of eIF-2α in cortical neurons. Due to the sensitivity of intracellular Ca2+-sensitive fluorescent dyes to Zn2+, it is not yet possible to determine whether Zn2+ treatment also evokes a release of Ca2+ from intracellular stores and therefore whether Zn2+ acts by a mechanism similar to that of thapsigargin. However, in accordance with the involvement of a common process, the effects of Zn2+ and thapsigargin were not additive on both the increase in eIF-2α phosphorylation and the inhibition of protein synthesis. PERK activation could intervene in the unfolded-protein response, which consists of the attenuation of protein synthesis rate following the accumulation of incorrectly folded proteins in the endoplasmic reticulum (31Harding H.P. Zang Y. Ron D. Nature. 1999; 397: 271-274Crossref PubMed Scopus (2457) Google Scholar). As Zn2+ may alter the folding of newly synthesized peptides (11Vallee B.L. Falchuk K.H. Physiol. Rev. 1993; 73: 79-117Crossref PubMed Google Scholar), one can speculate that PERK is responsible for the increase in eIF-2α phosphorylation in response to Zn2+, a process that could prevent further accumulation of incorrectly folded proteins in the endoplasmic reticulum. However, the increase in eIF-2α phosphorylation measured in neurons exposed to Zn2+ may also result from the inhibition of phosphatase activity, as okadaic acid induced a similar increase in eIF-2α phosphorylation. As PERK (or PKR) activation involves an autophosphorylation process, one cannot conclude whether the okadaic acid effect results from the inhibition of the dephosphorylation of the kinase or of the initiation factor. A complex consisting of eIF-2α, GTP, and tRNAMet must be formed during each cycle of translation initiation. This requires the regeneration of active eIF-2α by exchange of an eIF-2α-bound GDP for GTP, a process catalyzed by eIF-2B. The affinity of eIF-2B for the phosphorylated α-subunit of eIF-2 is 150-fold greater than for the unphosphorylated form of the protein (33Rowlands A.G. Panniers R. Henshaw E. J. Biol. Chem. 1988; 263: 5526-5533Abstract Full Text PDF PubMed Google Scholar) and the level of eIF-2α in the brain is 5 times higher than that of eIF-2B (34Alcazar A. Martin M.E. Soria E. Rodriguez S. Fando J.L. Salinas M. J. Neurochem. 1995; 65: 754-761Crossref PubMed Scopus (19) Google Scholar). Therefore, one might expect that if only 20% of the total amount of eIF-2α is phosphorylated, most of eIF-2B should become unavailable to catalyze the guanine nucleotide exchange on the remaining unphosphorylated pool of eIF-2α. If the relative amounts of eIF-2α and eIF-2B are in the same range in the brain and in cortical neurons, a relatively low rate of phosphorylation of eIF-2α, as detected in cortical neurons, could account for the large inhibition of protein synthesis induced by Zn2+ treatment or following mobilization of Ca2+ from intracellular stores. One important issue concerns the physiological significance of the Zn2+-induced inhibition of protein synthesis. The transient inhibition of protein translation in neurons exposed to Zn2+ might lead to the expression of a new pattern of proteins that can be involved in specific pathological or physiological processes (35Ryazanov A.G. Spirin A.S. Translational Regulation of Gene Expression. Plenum Press, New York1993: 433-455Google Scholar, 36Palfrey H.C. Nairn A.C. Adv. Second Messenger Phosphoprotein Res. 1995; 30: 191-223Crossref PubMed Scopus (35) Google Scholar). Apoptosis, but not necrosis, is an active process requiring protein synthesis, which is thus suppressed by protein synthesis inhibitors. Accordingly, we have demonstrated that the inhibition of protein synthesis by cycloheximide or diphtheria toxin treatments (which are not toxic by themselves) protects cortical neurons from the toxicity evoked by low concentrations of NMDA (15Marin P. Nastiuk K.L. Daniel N. Girault J.A. Czernik A.J. Glowinski J. Nairn A.C. Prémont J. J. Neurosci. 1997; 17: 3445-3454Crossref PubMed Google Scholar), which are known to selectively induce an apoptotic process (37Bonfoco E. Krainc D. Ankarcrona M. Nicotera P. Lipton S.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7162-7166Crossref PubMed Scopus (1855) Google Scholar). On the contrary, the pharmacological inhibition of protein synthesis did not protect neurons against strong excitotoxicity (37Bonfoco E. Krainc D. Ankarcrona M. Nicotera P. Lipton S.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7162-7166Crossref PubMed Scopus (1855) Google Scholar). Therefore, the inhibition of protein synthesis seems to constitute a self-protecting mechanism rather than an active deleterious process. However, due to the marked inhibition of protein synthesis induced by Zn2+, which is in the same range as those evoked by cycloheximide and diphtheria toxin treatments, it was impossible to demonstrate such a protective mechanism in Zn2+-induced neurotoxicity. Translational control in neurons could contribute to long term synaptic plasticity such as long term potentiation (LTP). The application of protein synthesis inhibitors during the induction of LTP in the hippocampus reduces its duration to 3–6 h, indicating that a critical level of protein synthesis is required for long term occurrence of LTP (38Bliss T.V.P. Collingridge G.L. Nature. 1993; 361: 31-39Crossref PubMed Scopus (9396) Google Scholar). The combined phosphorylation of eIF-2α and eEF-2 following Zn2+ and glutamate release during LTP induction could lead to a transient inhibition of protein synthesis, allowing the establishment of a new pattern of protein expression required for the maintenance of LTP. Therefore, the phosphorylation of eIF-2α by Zn2+ and of eEF-2 by glutamate and their convergent consequences on protein translation open new perspectives for understanding the mechanisms implicated in such an unusual co-transmission process. We thank Dr. Christopher Proud for kindly providing us with the monoclonal eIF-2α antibody.

Almost a billion people worldwide are chronically undernourished. Herein, using a mouse model of coxsackievirus B3 (CVB3) infection, we report that a single day of food restriction (FR) markedly increases susceptibility to attenuated enterovirus infection, replication, and disease. These "pro-viral" effects, which are rapidly-reversed by the restoration of food, are mediated by several genes whose expression is altered by FR, and which support CVB3 replication. Central to this is TFEB, a protein whose expression and activation status are rapidly increased by FR. TFEB, which regulates the transcription of >100 genes involved in macroautophagy/autophagy and lysosomal biogenesis, responds similarly to both FR and CVB3 infection and plays a pivotal role in determining host susceptibility to CVB3. We propose that, by upregulating TFEB, FR generates an intracellular environment that is more hospitable to the incoming virus, facilitating its replication. This interplay between nutritional status and enterovirus replication has implications for human health and, perhaps, for the evolution of these viruses.Abbreviations: Atg/ATG: autophagy-related; CAR: Coxsackievirus and adenovirus receptor; Cas9: CRISPR associated protein 9; Cre: recombinase that causes recombination; CRISPR: clustered regularly interspaced short palindromic repeats; Ctsb/CTSB: cathepsin B; CVB3: coxsackievirus B3; DsRedCVB3: a recombinant CVB3 that encodes the Discosoma red fluorescent protein; EL: elastase; FR: food restriction; GFP: green fluorescent protein; gRNA: guide RNA; HBSS: Hanks Buffered Salt Solution; LYNUS: lysosomal nutrient sensing machinery; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; Nluc: nanoluciferase; NlucCVB3: a recombinant CVB3 encoding nanoluciferase; pfu: plaque-forming unit(s); p.i.: post infection; rCVB: recombinant coxsackievirus B3; RPS6KB/p70S6K: ribosomal protein S6 kinase; RT: room temperature; siRNA: small interfering RNA; TFEB: transcription factor EB; tg: transgenic; TUBB: β-tubulin; UNINF: uninfected; wrt: with respect to; WT: wild type.

An effective HIV vaccine likely requires the elicitation of neutralizing antibodies (NAbs) against multiple HIV-1 clades. The recently developed cleavage-independent native flexibly linked (NFL) envelope (Env) trimers exhibit well-ordered conformation and elicit autologous tier 2 NAbs in multiple animal models. Here, we investigated whether the fusion of molecular adjuvant C3d to the Env trimers can improve B- cell germinal center (GC) formation and antibody responses. To generate Env-C3d trimers, we performed a glycine-serine- based (G 4 S) flexible peptide linker screening and identified a linker range that allowed native folding. A 30–60- amino- acid- long linker facilitates Env-to-C3d association and achieves the secretion of well-ordered trimers and the structural integrity and functional integrity of Env and C3d. The fusion of C3d did not dramatically affect the antigenicity of the Env trimers and enhanced the ability of the Env trimers to engage and activate B cells in vitro . In mice, the fusion of C3d enhanced germinal center formation, the magnitude of Env-specific binding antibodies, and the avidity of the antibodies in the presence of an adjuvant. The Sigma Adjuvant System (SAS) did not affect the trimer integrity in vitro but contributed to altered immunogenicity in vivo , resulting in increased tier 1 neutralization, likely by increased exposure of variable region 3 (V3). Taken together, the results indicate that the fusion of the molecular adjuvant, C3d, to the Env trimers improves antibody responses and could be useful for Env-based vaccines against HIV.

The contraction phase of the T cell response is a poorly understood period after the resolution of infection when virus-specific effector cells decline in number and memory cells emerge with increased frequencies. CD8(+) T cells plummet in number and quickly reach stable levels of memory following acute lymphocytic choriomeningitis virus infection in mice. In contrast, virus-specific CD4(+) T cells gradually decrease in number and reach homeostatic levels only after many weeks. In this study, we provide evidence that MHCII-restricted viral Ag persists during the contraction phase following this prototypical acute virus infection. We evaluated whether the residual Ag affected the cell division and number of virus-specific naive and memory CD4(+) T cells and CD8(+) T cells. We found that naive CD4(+) T cells underwent cell division and accumulated in response to residual viral Ag for >2 mo after the eradication of infectious virus. Surprisingly, memory CD4(+) T cells did not undergo cell division in response to the lingering Ag, despite their heightened capacity to recognize Ag and make cytokine. In contrast to CD4(+) T cells, CD8(+) T cells did not undergo cell division in response to the residual Ag. Thus, CD8(+) T cells ceased division within days after the infection was resolved, indicating that CD8(+) T cell responses are tightly linked to endogenous processing of de novo synthesized virus protein. Our data suggest that residual viral Ag delays the contraction of CD4(+) T cell responses by recruiting new populations of CD4(+) T cells.

Zinc released from a subpopulation of glutamatergic synapses, mainly localized in the cerebral cortex and the hippocampus, facilitates or reduces glutamatergic transmission by acting on neuronal AMPA and NMDA receptors, respectively. However, neurons are not the only targets of zinc. In the present study, we provide evidence that zinc inhibits protein synthesis in cultured astrocytes from the cerebral cortex of embryonic mice. This inhibition, which reached 85% in the presence of 100 micro m zinc, was partially and slowly reversible and resulted from the successive inhibition of the elongation and the initiation steps of the protein translation process. This was assessed by measuring the phosphorylation level of the elongation factor eEF-2 and of the alpha subunit of the initiation factor eIF-2. Due to the rapid turnover of connexin-43 that forms junction channels in cultured astrocytes, the zinc-induced decrease of protein synthesis led to a partial disappearance of connexin-43, which was associated with an inhibition of the cellular coupling in the astrocytic syncitium. In conclusion, zinc not only inhibits protein synthesis in neurons, as previously demonstrated, but also in astrocytes. The resulting decrease in the intercellular communication between astrocytes should alter the function of surrounding neurons as well as their survival.

In vivo interactions between RNA viruses and autophagy were first postulated several decades ago. For example, in the 1970s, coxsackievirus was shown to cause the accumulation of autophagic vacuoles in the pancreatic acinar cells of infected mice and, in 1986, activation of hepatic autophagy in influenza B virus-infected mice was reported. However, detailed analyses awaited a fuller understanding of the autophagy pathway and its constituent proteins. In this chapter, we describe studies of autophagy and RNA viruses that have used in vivo analysis in both invertebrates and vertebrates.

Almost a billion people worldwide are chronically undernourished. Herein, using a mouse model of coxsackievirus B3 (CVB3) infection, we report that a single day of food restriction (FR) markedly increases susceptibility to attenuated enterovirus infection, replication, and disease. These “pro-viral” effects, which are rapidly-reversed by the restoration of food, are mediated by several genes whose expression is altered by FR, and which support CVB3 replication. Central to this is TFEB, a protein whose expression and activation status are rapidly increased by FR. TFEB, which regulates the transcription of &gt;100 genes involved in macroautophagy/autophagy and lysosomal biogenesis, responds similarly to both FR and CVB3 infection and plays a pivotal role in determining host susceptibility to CVB3. We propose that, by upregulating TFEB, FR generates an intracellular environment that is more hospitable to the incoming virus, facilitating its replication. This interplay between nutritional status and enterovirus replication has implications for human health and, perhaps, for the evolution of these viruses. <b>Abbreviations:</b> Atg/ATG: autophagy-related; CAR: Coxsackievirus and adenovirus receptor; Cas9: CRISPR associated protein 9; Cre: recombinase that causes recombination; CRISPR: clustered regularly interspaced short palindromic repeats; <i>Ctsb</i>/CTSB: cathepsin B; CVB3: coxsackievirus B3; DsRedCVB3: a recombinant CVB3 that encodes the Discosoma red fluorescent protein; EL: elastase; FR: food restriction; GFP: green fluorescent protein; gRNA: guide RNA; HBSS: Hanks Buffered Salt Solution; LYNUS: lysosomal nutrient sensing machinery; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; Nluc: nanoluciferase; NlucCVB3: a recombinant CVB3 encoding nanoluciferase; pfu: plaque-forming unit(s); p.i.: post infection; rCVB: recombinant coxsackievirus B3; RPS6KB/p70S6K: ribosomal protein S6 kinase; RT: room temperature; siRNA: small interfering RNA; TFEB: transcription factor EB; tg: transgenic; TUBB: β-tubulin; UNINF: uninfected; wrt: with respect to; WT: wild type.

Les trois principaux agents neurotoxiques liberes et/ou produits apres une reperfusion qui suit un episode isquemique provoquent une inhibition importante de la synthese proteique. Nous avons precedemment montre que le glutamate, le principal neurotransmetteur dans le cerveau, provoque une augmentation du Ca2+ cytosolique responsable du blocage de la synthese proteique au niveau de l'etape d'elongation. Au cours de ce travail nous avons montre que le Zn2+ inhibe profondement la synthese proteique, induit une phosphorylation importante de eIF-2α et decroit le taux de polyribosomes suggerant que le Zn2+ bloque l'etape d'initiation. En revanche H2O2 provoque phosphorylation successive du facteur d'elongation, eEF-2 puis celle du facteur d'initiation eIF-2α, cette derniere apparaissant apres des temps long > 15 min ou a des concentrations elevee > 150 ưM. . .