Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells

The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium homeostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Surprisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.     

The autophagic response to nutrient deprivation in the hl-1 cardiac myocyte is modulated by Bcl-2 and sarco/endoplasmic reticulum calcium stores

Macroautophagy is a vital process in the cardiac myocyte: it plays a protective role in the response to ischemic injury, and chronic perturbation is causative in heart disease. Recent findings evidence a link between the apoptotic and autophagic pathways through the interaction of the antiapoptotic proteins Bcl-2 and Bcl-XL with Beclin 1. However, the nature of the interaction, either in promoting or blocking autophagy, remains unclear. Here, using a highly sensitive, macroautophagy-specific flux assay allowing for the distinction between enhanced autophagosome production and suppressed autophagosome degradation, we investigated the control of Beclin 1 and Bcl-2 on nutrient deprivation-activated macroautophagy. We found that in HL-1 cardiac myocytes the relationship between Beclin 1 and Bcl-2 is subtle: Beclin 1 mutant lacking the Bcl-2-binding domain significantly reduced autophagic activity, indicating that Beclin 1-mediated autophagy required an interaction with Bcl-2. Overexpression of Bcl-2 had no effect on the autophagic response to nutrient deprivation; however, targeting Bcl-2 to the sarco/endoplasmic reticulum (S/ER) significantly suppressed autophagy. The suppressive effect of S/ER-targeted Bcl-2 was in part due to the depletion of S/ER calcium stores. Intracellular scavenging of calcium by BAPTA-AM significantly blocked autophagy, and thapsigargin, an inhibitor of sarco/endoplasmic reticulum calcium ATPase, reduced autophagic activity by approximately 50%. In cells expressing Bcl-2-ER, thapsigargin maximally reduced autophagic flux. Thus, our results demonstrate that Bcl-2 negatively regulated the autophagic response at the level of S/ER calcium content rather than via direct interaction with Beclin 1. Moreover, we identify calcium homeostasis as an essential component of the autophagic response to nutrient deprivation.     

Brefeldin A, thapsigargin, and AIF4- stimulate the accumulation of GRP78 mRNA in a cycloheximide dependent manner, whilst induction by hypoxia is independent of protein synthesis.

The glucose regulated proteins (GRPs) are major structural components of the endoplasmic reticulum (ER) and are involved in the import, folding, and processing of ER proteins. Expression of the glucose regulated proteins (GRP78 and GRP94) is greatly increased after cells are exposed to stress agents (including A23187 and tunicamycin) which inhibit ER function. Here, we demonstrate that three novel inhibitors of ER function, thapsigargin (which inhibits the ER Ca(2+)-ATPase), brefeldin A (an inhibitor of vesicle transport between the ER and Golgi) and AIF4-, (which inhibits trimeric G-proteins), can increase the expression of both GRP78 and 94. The common characteristic shared by activators of GRP expression is that they disrupt some function of the ER. The increased levels of GRPs may be a response to the accumulation of aberrant proteins in the ER or they may be increased in response to structural/functional damage to the ER. The increased accumulation of GRP78 mRNA after exposure of cells to either thapsigargin, brefeldin A, AIF4-, A23187, or tunicamycin can be blocked by pre-incubation in cycloheximide. In contrast, accumulation of GRPs after exposure to hypoxia was independent of cycloheximide. In addition, the protein kinase inhibitor genistein blocked the thapsigargin induced accumulation of GRP78 mRNA, whereas the protein phosphatase inhibitor okadaic acid caused increased accumulation of GRP78 mRNA. The data indicates that there are at least 2 mechanisms for induced expression of GRPs, one of which involves a phosphorylation step and requires new protein synthesis (e.g., thapsigargin, A23187) and one which is independent of both these steps (hypoxia).     

UAE1 inhibition mediates the unfolded protein response,DNA damage and caspase-dependent cell death in pancreatic cancer

The Unfolded Protein Response (UPR) plays a key role in the adaptive response to loss of protein homeostasis within the endoplasmic reticulum (ER). The UPR has an adaptive function in protein homeostasis, however, sustained activation of the UPR due to hypoxia, nutrient deprivation, and increased demand for protein synthesis, alters the UPR program such that additional perturbation of ER homeostasis activates a pro-apoptotic program. Since ubiquitination followed by proteasomal degradation of misfolded proteins within the ER is a central mechanism for restoration of ER homeostasis, inhibitors of this pathway have proven to be valuable anti-cancer therapeutics. Ubiquitin activating enzyme 1(UAE1), activates ubiquitin for transfer to target proteins for proteasomal degradation in conjunction with E2 and E3 enzymes. Inhibition of UAE1 activity in response to TAK-243, leads to an accumulation of misfolded proteins within the ER, thereby aggravating ER stress, leading to DNA damage and arrest of cells in the G2/M phase of the cell cycle. Persistent drug treatment mediates a robust induction of apoptosis following a transient cell cycle arrest. These biological effects of TAK-243 were recapitulated in mouse models of PDAC demonstrating antitumor activity at a dose and schedule that did not exhibit obvious normal tissue toxicity. In vitro as well as studies in mouse models failed to show enhanced efficacy when TAK-243 was combined with ionizing radiation or gemcitabine, providing an impetus for future studies to identify agents that synergize with this class of agents for improved tumor control in PDAC.SignificanceThe UAE1 inhibitor TAK-243, mediates activation of the unfolded protein response, accumulation of DNA breaks and apoptosis, providing a rationale for the use as a safe and efficacious anti-cancer therapeutic for PDAC.     

The ubiquitin E3 ligase POSH regulates calcium homeostasis through spatial control of Herp

The ubiquitin (Ub) domain protein Herp plays a crucial role in the maintenance of calcium homeostasis during endoplasmic reticulum (ER) stress. We now show that Herp is a substrate as well as an activator of the E3 Ub ligase POSH. Herp-mediated POSH activation requires the Ubl domain and exclusively promotes lysine-63-linked polyubiquitination. Confocal microscopy demonstrates that Herp resides mostly in the trans-Golgi network, but, shortly after calcium perturbation by thapsigargin (Tpg), it appears mainly in the ER. Substitution of all lysine residues within the Ubl domain abolishes lysine-63-linked polyubiquitination of Herp in vitro and calcium-induced Herp relocalization that is also abrogated by the overexpression of a dominant-negative POSHV14A. A correlation exists between the kinetics of Tpg-induced Herp relocalization and POSH-dependent polyubiquitination. Finally, the overexpression of POSH attenuates, whereas the inhibition of POSH by the expression of POSHV14A or by RNA interference enhances Tpg-induced calcium burst. Altogether, these results establish a critical role for POSH-mediated ubiquitination in the maintenance of calcium homeostasis through the spatial control of Herp.     

SERCaMP: a carboxy-terminal protein modification that enables monitoring of ER calcium homeostasis

Endoplasmic reticulum (ER) calcium homeostasis is disrupted in diverse pathologies, including neurodegeneration, cardiovascular diseases, and diabetes. Temporally defining calcium dysregulation during disease progression, however, has been challenging. Here we describe secreted ER calcium-monitoring proteins (SERCaMPs), which allow for longitudinal monitoring of ER calcium homeostasis. We identified a carboxy-terminal modification that is sufficient to confer release of a protein specifically in response to ER calcium depletion. A Gaussia luciferase (GLuc)–based SERCaMP provides a simple and sensitive method to monitor ER calcium homeostasis in vitro or in vivo by analyzing culture medium or blood. GLuc-SERCaMPs revealed ER calcium depletion in rat primary neurons exposed to various ER stressors. In vivo, ER calcium disruption in rat liver was monitored over several days by repeated sampling of blood. Our results suggest that SERCaMPs will have broad applications for the long-term monitoring of ER calcium homeostasis and the development of therapeutic approaches to counteract ER calcium dysregulation.     

The unfolded protein response in melanocytes: activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding

Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.     

The cargo receptor ERGIC-53 is a target of the unfolded protein response

The accumulation of unfolded proteins in the ER triggers a signaling response known as unfolded protein response (UPR). In yeast the UPR affects several hundred genes that encode ER chaperones and proteins operating at later stages of secretion. In mammalian cells the UPR appears to be more limited to chaperones of the ER and genes assumed to be important after cell recovery from ER stress that are not important for secretion. Here, we report that the mRNA of lectin ERGIC-53, a cargo receptor for the transport of glycoproteins from ER to ERGIC, and of its related protein VIP36 is induced by the known inducers of ER stress, tunicamycin and thapsigargin. In parallel, the rate of synthesis of the ERGIC-53 protein was induced by these agents. The response was due to the UPR since it was also triggered by castanospermine, a specific inducer of UPR, and inhibited by genistein. Thapsigargin-induced upregulation of ERGIC-53 could be fully accounted for by the ATF6 pathway of UPR. The results suggest that in mammalian cells the UPR also affects traffic from and beyond the ER.     

Effect of mefloquine on the mechanical activity of the mouse isolated rectal smooth muscle.

The effects of mefloquine on the mechanical activity of the mouse isolated rectal smooth muscle was studied. Mefloquine (4.1x10-5 - 5.2x10-3M) when applied alone and separately exerted variable effects on the rectum. In some preparations, it caused slight phasic contractions while in others no response was elicited. When the external Ca(2+) was increased from 1.8mM to 300mM mefloquine produced phasic contractile activity which was abolished on return to normal 1.8mM suggesting that the contractile activity was due to extracellular Ca(2+) influx. Meflaquine [4.1x10-6M - 4.1x10-4M] caused contraction - dependent inhibition of KCL, Carbachol and CaCl2 [in depolarizing Tyrode Solution]. Mefloquine [2.1x10-4M] blocked KCL, but not carbachol contractions which were largely reversed by increasing [Ca2+]. The results show that mefloquine possesses anticholinergic and appreciable calcium channel blocking activity.     

Regulation of calcium signalling by docosahexaenoic acid in human T-cells. Implication of CRAC channels

We elucidated the role of docosahexaenoic acid (DHA) on the increases in free intracellular calcium concentrations, [Ca(2+)]i, in human (Jurkat) T-cell lines. DHA evoked an increase in [Ca(2+)]i in a dose-dependent manner in these cells. Anti-CD3 antibody, known to stimulate increases in Ca(2+) from endoplasmic reticulum (ER) via the production of inositol trisphosphate, also evoked increases in [Ca(2+)]i in Jurkat T-cells. We also used thapsigargin which inhibits Ca(2+)-ATPase of the ER and, therefore, increases Ca(2+) in the cytosol. Interestingly, addition of DHA during the thapsigargin-induced peak response exerted an additive effect on the increases in [Ca(2+)]i in human T-cells, indicating that the mechanisms of action of these two agents are different. However, the DHA-induced calcium response was not observed when this agent was added during the anti-CD3-induced calcium peak, though its addition resulted in a prolonged and sustained calcium response as a function of time, suggesting that DHA recruits calcium, in part, from the ER pool and the prolonged response may be due to Ca(2+) influx. In the medium containing 0% Ca(2+), the DHA-evoked response on the increases in [Ca(2+)]i was significantly curtailed as compared to that in 100% Ca(2+) medium, supporting the notion that the response of the DHA is also due, in part, to the opening of calcium channels. Furthermore, preincubation of cells with tyrphostin A9, an inhibitor of Ca(2+) release-activated Ca(2+) (CRAC) channels also significantly curtailed the DHA-induced sustained response on the increases in [Ca(2+)]i in these cells. These results suggest that DHA induces an increase in [Ca(2+)]i via the ER pool and the opening of CRAC channels in human T-cells.     

小鼠脑组织及培养神经元Neuro-2a 在内质网应激反应时的基因表达谱分析

内质网是真核细胞的重要细胞器。某些细胞内外因素如病原体感染等能引起从内质网到胞浆和胞核的信号传导途径活化,即内质网应激反应。但是,目前国内外尚无针对内质网应激反应的基因表达谱分析报道。本研究中,用3种已报道的内质网应激反应诱导剂,包括蛋白质糖基化抑制剂衣霉素(tunicamycin)、内质网Ca ²⁺-ATPases抑制剂毒胡萝卜素（thapsigargin）和乙脑病毒(Japanese encephalitis virus, JEV),分别处理小鼠颅腔和小鼠脑神经瘤细胞（Neuro-2a）,试剂处理组与未处理组的第二代RNA测序分析发现,衣霉素、毒胡萝卜素和乙脑病毒在体外和体内均引起分子伴侣基因Hsp70表达上调,诱导内质网应激反应。衣霉素、毒胡萝卜素和乙脑病毒体外处理诱导的内质网应激反应信号通路中,基因差异表达相似性高于体内处理组。乙脑病毒和糖基化抑制剂衣霉素体内外处理,主要诱导内质网应激反应的非折叠蛋白质反应信号通路,引起相关基因Atf4、Bip、Edem和Perk等表达上调。内质网Ca ²⁺-ATPases抑制剂毒胡萝卜素主要诱导内质网超负荷反应,激活NF-κB信号通路。乙脑病毒诱导的内质网应激反应相关差异表达基因数量最多,体外与体内合计有40种。乙脑病毒体内外处理上调的基因包括Bax、Casp12、Atf4、Bip、Edem和Perk等,下调的基因包括Sec23/24、Nef、Svip和Jnk等。糖基化抑制剂衣霉素体内外处理上调基因包括Gadd34、Atf4、Ermani和Bip等,下调基因包括Grp94、Atf6、Sec23/24和Nef等。内质网Ca ^-2+-ATPases抑制剂毒胡萝卜素体内外处理上调的基因包括Sec61、Trap和Ask1等。衣霉素、毒胡萝卜素和乙脑病毒体内外处理也通过内质网应激反应,调控与炎症或凋亡相关的MAPK信号通路和P53信号通路。本研究首次通过使用3种内质网应激反应诱导剂分别处理小鼠和细胞,揭示了体内外内质网应激反应引起的基因表达谱变化,为内质网应激反应相关疾病的治疗提供了新思路。     

Impact of serine protease inhibitor alpha1-antitrypsin on expression of endoplasmic reticulum stress-induced proinflammatory factors in adipocytes

Obesity-induced endoplasmic reticulum (ER) stress contributes to low-grade chronic inflammation in adipose tissue and may cause metabolic disorders such as diabetes mellitus and dyslipidemia. Identification of high serpina A1 (alpha-1 antitrypsin, A1AT) expression in mouse adipose tissue and adipocytes prompted us to explore the role of A1AT in the inflammatory response of adipocytes under ER stress. We aimed to determine the role of A1AT expression in adipocytes with ER stress during regulation of adipocyte homeostasis and inflammation. To this end, we chemically induced ER stress in A1AT small interfering RNA-transfected differentiating adipocytes using thapsigargin. Induction of CCAAT-enhancer-binding protein homologous protein (CHOP), an ER stress marker, by thapsigargin was lower in A1AT-deficient SW872 adipocytes. Thapsigargin or the proinflammatory cytokine tumor necrosis factor (TNF)α increased basal expression of cytokines such as interleukin (IL)-1β and IL-8 in both SW872 and primary omental adipocytes. This thapsigargin- or TNFα-induced expression of proinflammatory genes was increased by A1AT deficiency. These findings indicate that adipose A1AT may suppress the ER stress response to block excessive expression of proinflammatory factors, which suggests that A1AT protects against adipose tissue dysfunction associated with ER stress activation.     

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress—the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (TG)—potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.     

Use of mefloquine in children - a review of dosage,pharmacokinetics and tolerability data

Background

Use of anti-malarial medication in children is hampered by a paucity of dosage, pharmacokinetic and tolerability data.

Methods

Data on the use of mefloquine in children, particularly in young children weighing less than 20 kg, were reviewed using PubMed literature and reports on file.

Results

Chemoprophylaxis data: Two studies with a total of 170 children were found. A simulated mefloquine plasma profile showed that doses to achieve protective chemoprophylaxis blood concentration of mefloquine of approximately 620 ng/mL (or 1.67 μmol/L) in children should be at least 5 mg/kg. This simulated plasma profile in children corresponds to that seen in adult travellers using a weekly prophylaxis dose of 250 mg. This reinforces current practice of using weight-based dosage for children. Clearance per body weight is higher in older children. For children who travel to malaria risk areas tablets can be broken and crushed as required. It is necessary to disguise the bitter taste of the drug.Treatment data: Mefloquine treatment (alone or in combination) data are available for more than 6000 children of all age and weight categories. The stereoselectivity and pharmacokinetic profile of mefloquine in children is similar to that observed in adults. There is higher clearance in older children (aged 5-12 years) compared to younger children (aged 6-24 months). Mefloquine treatment is well tolerated in infants (5-12 kg) but vomiting is a problem at high doses. This led to the use of a "split dose" regimen with 15 mg/kg initially, followed 12 hours later by 10 mg/kg.Mefloquine 125 mg has been used as intermittent preventive treatment (IPT) and was found to be efficacious in reducing episodes of malaria in a moderate-transmission setting but vomiting was a problem in 8% of children aged 2-11 months.Mefloquine is also used as a component of artemisinin combination therapy (ACT) in small children. The combination artesunate plus mefloquine is a WHO approved first-line treatment for uncomplicated malaria in Africa.

Conclusion

Currently available data provide a scientific basis for the use of mefloquine in small children in the chemoprophylaxis setting and as a part of treatment regimens for children living in endemic areas.