The unfolded protein response regulates glutamate receptor export from the endoplasmic reticulum

Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors mediate the majority of excitatory signaling in the CNS, and the functional properties and subcellular fate of these receptors depend on receptor subunit composition. Subunit assembly is thought to occur in the endoplasmic reticulum (ER), although we are just beginning to understand the underlying mechanism. Here we examine the trafficking of Caenorhabditis elegans glutamate receptors through the ER. Our data indicate that neurons require signaling by the unfolded protein response (UPR) to move GLR-1, GLR-2, and GLR-5 subunits out of the ER and through the secretory pathway. In contrast, other neuronal transmembrane proteins do not require UPR signaling for ER exit. The requirement for the UPR pathway is cell type and age dependent: impairment for receptor trafficking increases as animals age and does not occur in all neurons. Expression of XBP-1, a component of the UPR pathway, is elevated in neurons during development. Our results suggest that UPR signaling is a critical step in neural function that is needed for glutamate receptor assembly and secretion.     

HIV-1 Tat causes apoptotic death and calcium homeostasis alterations in rat neurons

We investigated the role of the HIV-1 protein Tat in AIDS-associated dementia, by studying its toxicity on rat cortical and hippocampal neurons in vitro. We evaluated the involvement of astroglial cells and of caspase transduction pathway in determining Tat toxicity. Here we report that synthetic Tat(1-86) induced apoptotic death on cultured rat neurons in a time-dependent manner that was not influenced by glial coculture, and that was abolished by blocking caspase transduction pathway. A microfluorimetric analysis on the Tat excitatory properties on neurons, and its effect on intracellular calcium concentrations, revealed that Tat(1-86) induced increase in cytoplasmic free calcium concentrations in rat hippocampal and cortical neurons. This effect required extracellular calcium and was differently reduced by voltage dependent calcium channel blockers and both NMDA and non-NMDA glutamate receptors antagonists. Furthermore, we observed that Tat(1-86)-treated neurons showed increased sensitivity to the glutamate excitotoxicity. Thus, the Tat-induced neuronal injury seems to occur through a direct interaction of the protein with neurons, requires activation of caspases, and is likely to derive from Tat(1-86)-induced calcium loads and disruption of glutamatergic transmission.     

Calcium signaling at the endoplasmic reticulum: fine-tuning stress responses

Endoplasmic reticulum (ER) calcium signaling is implicated in a myriad of coordinated cellular processes. The ER calcium content is tightly regulated as it allows a favorable environment for protein folding, in addition to operate as a major reservoir for fast and specific release of calcium. Altered ER homeostasis impacts protein folding, activating the unfolded protein response (UPR) as a rescue mechanism to restore proteostasis. ER calcium release impacts mitochondrial metabolism and also fine-tunes the threshold to undergo apoptosis under chronic stress. The global coordination between UPR signaling and energetic demands takes place at mitochondrial associated membranes (MAMs), specialized subdomains mediating interorganelle communication. Here we discuss current models explaining the functional relationship between ER homeostasis and various cellular responses to coordinate proteostasis and metabolic maintenance.     

Cell death in HIV dementia

Many patients infected with human immunodeficiency virus type-1 (HIV-1) suffer cognitive impairment ranging from mild to severe (HIV dementia), which may result from neuronal death in the basal ganglia, cerebral cortex and hippocampus. HIV-1 does not kill neurons by infecting them. Instead, viral proteins released from infected glial cells, macrophages and/or stem cells may directly kill neurons or may increase their vulnerability to other cell death stimuli. By binding to and/or indirectly activating cell surface receptors such as CXCR4 and the N-methyl-D-aspartate receptor, the HIV-1 proteins gp120 and Tat may trigger neuronal apoptosis and excitotoxicity as a result of oxidative stress, perturbed cellular calcium homeostasis and mitochondrial alterations. Membrane lipid metabolism and inflammation may also play important roles in determining whether neurons live or die in HIV-1-infected patients. Drugs and diets that target oxidative stress, excitotoxicity, inflammation and lipid metabolism are in development for the treatment of HIV-1 patients.     

Differential Induction of Interleukin-10 in Monocytes by HIV-1 Clade B and Clade C Tat Proteins

The clade B human immunodeficiency virus, type 1 (HIV-1) Tat (trans-acting regulatory protein) induces interleukin-10 (IL-10) production in monocytes. IL-10, an anti-inflammatory cytokine, down-regulates proinflammatory cytokines and suppresses the immune response, leading to a rapid progression from HIV-1 infection to AIDS. Nine clades of HIV-1 are responsible for the majority of infections worldwide. Recent studies demonstrate that different HIV-1 clades have biological differences in relation to transmission, replication, and disease progression. In this study, we show that the cysteine to serine mutation at position 31, found in >90% of HIV-1 clade C Tat proteins, results in a marked decrease in IL-10 production in monocytes compared with clade B Tat. Additionally, the C31S mutation found in C Tat is responsible for the inability of these Tat proteins to produce high IL-10 levels in monocytes due to its inability to induce intracellular calcium flux through L-type calcium channels. Moreover, we show that p38α/p38β and phosphoinositide 3-kinase are crucial to Tat-induced IL-10 production. These findings provide further evidence that HIV-1 clades differ in their biological properties that may impact HIV-1 pathogenesis and disease progression.     

Human Immunodeficiency Virus Type 1 Tat Induces Apoptosis and Increases Sensitivity to Apoptotic Signals by Up-Regulating FLICE/Caspase-8

Apoptosis contributes to the loss of CD4 cells during human immunodeficiency virus type 1 (HIV-1) infection. Although the product of the env gene, gp160/gp120, is known to play a role in cell death mediated by HIV-1, the role of other HIV-1 genes in the process is unclear. We found that HIV-1 lacking the env gene (HIVΔenv) still induced apoptosis in T-cell lines and primary CD4 T cells. The ability to induce apoptosis was attributable to Tat, a viral regulatory protein. Tat induction of apoptosis was separate from the transactivation function of Tat, required expression of the second exon of Tat, and was associated with the increased expression and activity of caspase-8 (casp-8), a signaling molecule in apoptotic pathways. Moreover, induction of apoptosis could be prevented by treating cells with an inhibitor of casp-8. In addition, we show that HIV-1Δenv infection and Tat expression increased the sensitivity of cells to Fas-mediated apoptosis, an apoptotic pathway that signals via casp-8. The up-regulation of casp-8 by HIV-1 Tat expression may contribute to the increased apoptosis and sensitivity to apoptotic signals observed in the cells of HIV-1-infected persons.     

Direct effects of HIV-1 Tat on excitability and survival of primary dorsal root ganglion neurons: possible contribution to HIV-1-associated pain

The vast majority of people living with human immunodeficiency virus type 1 (HIV-1) have pain syndrome, which has a significant impact on their quality of life. The underlying causes of HIV-1-associated pain are not likely attributable to direct viral infection of the nervous system due to the lack of evidence of neuronal infection by HIV-1. However, HIV-1 proteins are possibly involved as they have been implicated in neuronal damage and death. The current study assesses the direct effects of HIV-1 Tat, one of potent neurotoxic viral proteins released from HIV-1-infected cells, on the excitability and survival of rat primary dorsal root ganglion (DRG) neurons. We demonstrated that HIV-1 Tat triggered rapid and sustained enhancement of the excitability of small-diameter rat primary DRG neurons, which was accompanied by marked reductions in the rheobase and resting membrane potential (RMP), and an increase in the resistance at threshold (R(Th)). Such Tat-induced DRG hyperexcitability may be a consequence of the inhibition of cyclin-dependent kinase 5 (Cdk5) activity. Tat rapidly inhibited Cdk5 kinase activity and mRNA production, and roscovitine, a well-known Cdk5 inhibitor, induced a very similar pattern of DRG hyperexcitability. Indeed, pre-application of Tat prevented roscovitine from having additional effects on the RMP and action potentials (APs) of DRGs. However, Tat-mediated actions on the rheobase and R(Th) were accelerated by roscovitine. These results suggest that Tat-mediated changes in DRG excitability are partly facilitated by Cdk5 inhibition. In addition, Cdk5 is most abundant in DRG neurons and participates in the regulation of pain signaling. We also demonstrated that HIV-1 Tat markedly induced apoptosis of primary DRG neurons after exposure for longer than 48 h. Together, this work indicates that HIV-1 proteins are capable of producing pain signaling through direct actions on excitability and survival of sensory neurons.     

Nox4-Derived H2O2 Mediates Endoplasmic Reticulum Signaling through Local Ras Activation

The unfolded-protein response (UPR) of the endoplasmic reticulum (ER) has been linked to oxidant production, although the molecular details and functional significance of this linkage are poorly understood. Using a ratiometric H₂O₂ sensor targeted to different subcellular compartments, we demonstrate specific production of H₂O₂ by the ER in response to the stressors tunicamycin and HIV-1 Tat, but not to thapsigargin or dithiothreitol. Knockdown of the oxidase Nox4, expressed on ER endomembranes, or expression of ER-targeted catalase blocked ER H₂O₂ production by tunicamycin and Tat and prevented the UPR following exposure to these two agonists, but not to thapsigargin or dithiothreitol. Tat also triggered Nox4-dependent, sustained activation of Ras leading to ERK, but not phosphatidylinositol 3-kinase (PI3K)/mTOR, pathway activation. Cell fractionation studies and green fluorescent protein (GFP) fusions of GTPase effector binding domains confirmed selective activation of endogenous RhoA and Ras on the ER surface, with ER-associated K-Ras acting upstream of the UPR and downstream of Nox4. Notably, the Nox4/Ras/ERK pathway induced autophagy, and suppression of autophagy unmasked cell death and prevented differentiation of endothelial cells in 3-dimensional matrix. We conclude that the ER surface provides a platform to spatially organize agonist-specific Nox4-dependent oxidative signaling events, leading to homeostatic protective mechanisms rather than oxidative stress.Coupled in part to its function as a major site of protein synthesis, the endoplasmic reticulum (ER) has emerged as an important signaling organelle, responding to various cell stresses and controlling cell fate. Much of this signaling is initiated on the ER membrane surface. In response to an overload of misfolded client proteins in the ER lumen, for example, transmembrane ER stress sensors such as IRE1, PERK, and ATF6 initiate signals on the cytosolic face of the ER to reduce global protein synthesis, promote protein folding, and increase the degradation of misfolded proteins (36). Failure of this response to alleviate protein misfolding stress leads to late expression of proteins such as CHOP, culminating in cell death. In addition to factors controlling this unfolded-protein response (UPR), cyclins, pro- and anti-apoptotic BH3 domain proteins, caspases, and signaling adapters associate with the ER surface and control cell cycle entry, cell death, Ca²⁺ flux, amino acid metabolism, oxidative-stress response, and autophagy (15, 17-19, 41). Thus, the ER integrates a variety of stresses (metabolic, protein misfolding, and oxidative) to coordinate cellular stress responses.Another ER transmembrane protein is the NADPH oxidase Nox4. Like other Nox family members, Nox4 produces H₂O₂ and is thought to function primarily in cell signaling. Consistent with its ER localization, Nox4 mediates oxidative inactivation of the ER-resident phosphatase PTP1B and responds to ER stress induced by the LDL oxysterol 7-ketocholesterol (10, 34). The presence of Nox4 on the ER surface is notable, since ER stress is associated with the production of reactive oxygen species (ROS), though the nature of this association is poorly understood. Agents that cause ER stress initiate ATF4-dependent glutathione (GSH) synthesis, which is necessary to diminish ROS production and subsequent cell death (15). The mechanism of ROS production is also unclear, but it is thought to be a consequence of the UPR, possibly downstream of CHOP (25, 26). For the most part, such studies suggest a role for oxidants as late effectors of cell death, downstream from the UPR. The ER itself may be a source of oxidants, as knockdown of the ER oxidase Ero-1 in pek-1-null worms diminishes oxidant production (15). To some extent, the excessive production of ROS by the stressed ER would seem to run counter to recent observations that chemical and physiologic ER stressors converge on the production of a hyperreduced ER interior (28). In addition, [rho⁰] Saccharomyces cerevisiae lacking mitochondria also fails to produce ROS in response to ER stress, suggesting a mitochondrial source (16). In part, such studies linking the ER and oxidative stress have been hampered by reliance on probes, such as dichlorofluorescein, that are untargeted and nonspecific, reacting with a broad variety of oxidants as well as non-ROS compounds, such as cytosolic cytochrome c.In this study, using an H₂O₂-specific probe targeted to the ER, we found that Nox4 participates early in ER stress signaling in an agonist-specific fashion through a novel process involving focal activation of Ras on the ER. This Nox4-dependent pathway leads to activation of autophagy, which prevents the progression of the UPR to cell death, thus distinguishing Nox4 oxidant signaling from oxidative stress.     

Snapin,Positive Regulator of Stimulation- Induced Ca2+ Release through RyR,Is Necessary for HIV-1 Replication in T Cells

To identify critical host factors necessary for human immunodeficiency virus 1 (HIV-1) replication, large libraries of short-peptide-aptamers were expressed retrovirally. The target of one inhibitor peptide, Pep80, identified in this screen was determined to be Snapin, a protein associated with the soluble N-ethyl maleimide sensitive factor adaptor protein receptor (SNARE) complex that is critical for calcium-dependent exocytosis during neurotransmission. Pep80 inhibited Ca²⁺ release from intracellular stores and blocked downstream signaling by direct interruption of the association between Snapin and an intracellular calcium release channel, the ryanodine receptor (RyR). NFAT signaling was preferentially abolished by Pep80. Expression of Snapin overcame Pep80-mediated inhibition of Ca²⁺/NFAT signaling and HIV-1 replication. Furthermore, Snapin induced HIV-1 replication in primary CD4⁺ T cells. Thus, through its interaction with RyR, Snapin is a critical regulator of Ca²⁺ signaling and T cell activation. Use of the genetically selected intracellular aptamer inhibitors allowed us to define unique mechanisms important to HIV-1 replication and T cell biology.     

IL-1β Signaling Promotes CNS-Intrinsic Immune Control of West Nile Virus Infection

West Nile virus (WNV) is an emerging flavivirus capable of infecting the central nervous system (CNS) and mediating neuronal cell death and tissue destruction. The processes that promote inflammation and encephalitis within the CNS are important for control of WNV disease but, how inflammatory signaling pathways operate to control CNS infection is not defined. Here, we identify IL-1β signaling and the NLRP3 inflammasome as key host restriction factors involved in viral control and CNS disease associated with WNV infection. Individuals presenting with acute WNV infection displayed elevated levels of IL-1β in their plasma over the course of infection, suggesting a role for IL-1β in WNV immunity. Indeed, we found that in a mouse model of infection, WNV induced the acute production of IL-1β in vivo, and that animals lacking the IL-1 receptor or components involved in inflammasome signaling complex exhibited increased susceptibility to WNV pathogenesis. This outcome associated with increased accumulation of virus within the CNS but not peripheral tissues and was further associated with altered kinetics and magnitude of inflammation, reduced quality of the effector CD8⁺ T cell response and reduced anti-viral activity within the CNS. Importantly, we found that WNV infection triggers production of IL-1β from cortical neurons. Furthermore, we found that IL-1β signaling synergizes with type I IFN to suppress WNV replication in neurons, thus implicating antiviral activity of IL-1β within neurons and control of virus replication within the CNS. Our studies thus define the NLRP3 inflammasome pathway and IL-1β signaling as key features controlling WNV infection and immunity in the CNS, and reveal a novel role for IL-1β in antiviral action that restricts virus replication in neurons.     

Human immunodeficiency virus type 1 subtype C Tat fails to induce intracellular calcium flux and induces reduced tumor necrosis factor production from monocytes

Over 50% of all human immunodeficiency virus type 1 (HIV-1) infections worldwide are caused by subtype C strains, yet most research to date focuses on subtype B, the subtype most commonly found in North America and Europe. The HIV-1 trans-acting regulatory protein (Tat) is essential for regulating productive replication of HIV-1. Tat is secreted by HIV-infected cells and alters several functions of uninfected bystander cells. One such function is that, by acting at the cell membrane, subtype B Tat stimulates the production of tumor necrosis factor (TNF) and chemokine (C-C motif) ligand 2 (CCL2) from human monocytes and can act as a chemoattractant. In this study, we show that the mutation of a cysteine to a serine at residue 31 of Tat commonly found in subtype C variants significantly inhibits the abilities of the protein to bind to chemokine (C-C motif) receptor 2 (CCR2), induce intracellular calcium flux, stimulate TNF and CCL2 production, and inhibit its chemoattractant properties. We also show that TNF is important in mediating some effects of extracellular Tat. This report therefore demonstrates the important functional differences between subtype C and subtype B Tat and highlights the need for further investigation into the different strains of HIV-1.     

Reduction of calcium release from the endoplasmic reticulum could only provide partial neuroprotection against beta-amyloid peptide toxicity

Beta-amyloid (Abeta) peptide has been suggested to play important roles in the pathogenesis of Alzheimer's disease (AD). Abeta peptide neurotoxicity was shown to induce disturbance of cellular calcium homeostasis. However, whether modulation of calcium release from the endoplasmic reticulum (ER) can protect neurons from Abeta toxicity is not clearly defined. In the present study, Abeta peptide-triggered ER calcium release in primary cortical neurons in culture is modulated by Xestospongin C, 2-aminoethoxydiphenyl borate or FK506. Our results showed that reduction of ER calcium release can partially attenuate Abeta peptide neurotoxicity evaluated by LDH release, caspase-3 activity and quantification of apoptotic cells. While stress signals associated with perturbations of ER functions such as up-regulation of GRP78 was significantly attenuated, other signaling machinery such as activation of caspase-7 transmitting death signals from ER to other organelles could not be altered. We further provide evidence that molecular signaling in mitochondria play also a significant role in determining neuronal apoptosis because Abeta peptide-triggered activation of caspase-9 was not significantly reduced by attenuating ER calcium release. Our results suggest that neuroprotective strategies aiming at reducing Abeta toxicity should include molecular targets linked to ER perturbations associated with ER calcium release as well as mitochondrial stress.