David M. Booth

Redox Nanodomains Are Induced by and Control Calcium Signaling at the ER-Mitochondrial Interface

The ER-mitochondrial interface is central to calcium signaling, organellar dynamics, and lipid biosynthesis. The ER and mitochondrial membranes also host sources and targets of reactive oxygen species (ROS), but their local dynamics and relevance remained elusive since measurement and perturbation of ROS at the organellar interface has proven difficult. Employing drug-inducible synthetic ER-mitochondrial linkers, we overcame this problem and demonstrate that the ER-mitochondrial interface hosts a nanodomain of H2O2, which is induced by cytoplasmic [Ca(2+)] spikes and exerts a positive feedback on calcium oscillations. H2O2 nanodomains originate from the mitochondrial cristae, which are compressed upon calcium signal propagation to the mitochondria, likely due to Ca(2+)-induced K(+) and concomitant water influx to the matrix. Thus, ER-mitochondrial H2O2 nanodomains represent a component of inter-organelle communication, regulating calcium signaling and mitochondrial activities.

Adiponectin Inhibits Tumor Necrosis Factor-α–Induced Vascular Inflammatory Response via Caveolin-Mediated Ceramidase Recruitment and Activation

Rationale: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. Objective: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin’s vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. Methods and Results: Adiponectin significantly reduced tumor necrosis factor (TNF)&agr;–induced intercellular adhesion molecule-1 expression and attenuated TNF&agr;-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin–induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNF&agr;-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNF&agr;-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. Conclusions: These results demonstrate for the first time that adiponectin inhibits TNF&agr;-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.

Reliance of ER-mitochondrial calcium signaling on mitochondrial EF-hand Ca2+ binding proteins. Miros, MICUs, LETM1 and solute carriers

Endoplasmic reticulum (ER) and mitochondria are functionally distinct with regard to membrane protein biogenesis and oxidative energy production, respectively, but cooperate in several essential cell functions, including lipid biosynthesis, cell signaling and organelle dynamics. The interorganellar cooperation requires local communication that can occur at the strategically positioned and dynamic associations between ER and mitochondria. Calcium is locally transferred from ER to mitochondria at the associations and exerts regulatory effects on numerous proteins. A common Ca(2+) sensing mechanism is the EF-hand Ca(2+) binding domain, many of which can be found in proteins of the mitochondria, including Miro1&2, MICU1,2&3, LETM1 and mitochondrial solute carriers. Recently, these proteins have triggered much interest and were described in reports with diverging conclusions. The present essay focuses on their shared features and established specific functions.

Subcellular ROS imaging methods: Relevance for the study of calcium signaling.

Recent advances in genetically encoded fluorescent probes have dramatically increased the toolkit available for imaging the intracellular environment. Perhaps the biggest improvements have been made in sensing specific reactive oxygen species (ROS) and redox changes under physiological conditions. The new generation of probes may be targeted to a wide range of subcellular environments. By targeting such probes to compartments and organelle surfaces they may be exposed to environments, which support local signal transduction and regulation. The close apposition of the endoplasmic reticulum (ER) with mitochondria and other organelles forms such a local environment where Ca(2+) dynamics are greatly enhanced compared to the bulk cytosol. We describe here how newly developed genetically encoded redox indicators (GERIs) might be used to monitor ROS and probe their interaction with Ca(2+) at both global and local level.

Adiponectin Inhibits Tumor Necrosis Factor- -Induced Vascular Inflammatory Response via Caveolin-Mediated Ceramidase Recruitment and Activation

Rationale: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. Objective: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin’s vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. Methods and Results: Adiponectin significantly reduced tumor necrosis factor (TNF)&agr;–induced intercellular adhesion molecule-1 expression and attenuated TNF&agr;-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin–induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNF&agr;-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNF&agr;-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. Conclusions: These results demonstrate for the first time that adiponectin inhibits TNF&agr;-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.

Adiponectin Inhibits TNF-α-Induced Vascular Inflammatory Response via Caveolin-Mediated Ceramidase Recruitment and Activation

Rationale: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. Objective: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin’s vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. Methods and Results: Adiponectin significantly reduced tumor necrosis factor (TNF)&agr;–induced intercellular adhesion molecule-1 expression and attenuated TNF&agr;-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin–induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNF&agr;-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNF&agr;-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. Conclusions: These results demonstrate for the first time that adiponectin inhibits TNF&agr;-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.

Abstract 2459: Chemotherapy resistance in pediatric neuroblastoma is associated with reduced ER-mitochondria tethering

Development of multidrug resistance poses a persistent problem in cancer treatment, yet its underlying causes remain obscure. A principal role for mitochondria has been sought as this organelle integrates diverse stress signals to impact cell fate. Endoplasmic reticulum (ER) and mitochondria (mito) interact at specialized coupling sites called mitochondria-associated ER membranes (MAMs). MAMs serve as micro-domains for the transfer of essential calcium and lipid signals to mitochondria and regulate apoptotic sensitivity. Tightening of ER-mito tethering constitutes an early response to cellular stress leading to apoptosis, and alterations in ER-mito tethering have been implicated in diabetes and neurodegeneration, suggesting that the deregulation of this process may have broad relevance in disease. Here, we define a novel mechanism for chemotherapy resistance due to selection for reduced ER-mito tethering. Most high-risk neuroblastoma patients initially respond to chemotherapy before relapsing with lethal therapy resistant disease acquired during the course of intensive multimodality treatment. We obtained isogenic neuroblastoma cell lines from the same 7 patients both at the time of diagnosis (chemosensitive) and at the time of relapse (chemoresistant). We evaluated mitochondrial biomass (citrate synthase activity), mtDNA content (qPCR), and mtDNA sequence (Affymetrix MitoChip v2.0) but identified no changes correlated with acquired resistance. Electron microscopy image analyses of ER-mito interfaces revealed that tumors at relapse contain up to 70% fewer ER-mito tether complexes than their matched at-diagnosis tumors, as confirmed by IB for organelle-specific proteins. Mitochondria isolated from all 7 post-relapse tumors show attenuated cytochrome c release in response to tBid and Bim BH3 peptide, terminal death effectors downstream of most therapeutic stress. This attenuated mitochondrial response can be phenocopied by limited protelolysis of mitochondria to reduce ER-mito tethers. Reduced mitochondrial apoptotic signaling in post-relapse tumors correlates directly with chemoresistance (up to 800-fold) across diverse agent classes. To functionally validate this relationship, Cyclosporine A (CsA), a cyclophilin D inhibitor, was used in tumors at diagnosis to reduce ER-mito tethering. This led to attenuated apoptotic responses in isolated mitochondria, and increased tumor cell IC50 to diverse chemotherapeutics, partially phenocopying the therapy resistant state. Our findings support a novel model of differential apoptotic signaling in therapy resistant cells that relies on the altered proximity and interactions of the mitochondria with ER that may be harnessed to design more effective anti-cancer drug therapies. Citation Format: Jorida Coku, Elizabeth O. Scadden, Kangning Liu, Annette Vu, David M. Booth, Michelle Chen, Sharon Kim, C. Patrick Reynolds, Gyorgy Hajnoczky, Michael D. Hogarty. Chemotherapy resistance in pediatric neuroblastoma is associated with reduced ER-mitochondria tethering. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2459.

Adiponectin Inhibits Tumor Necrosis Factor-α–Induced Vascular Inflammatory Response via Caveolin-Mediated Ceramidase Recruitment and ActivationNovelty and Significance

Rationale: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. Objective: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin’s vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. Methods and Results: Adiponectin significantly reduced tumor necrosis factor (TNF)&agr;–induced intercellular adhesion molecule-1 expression and attenuated TNF&agr;-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin–induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNF&agr;-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNF&agr;-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. Conclusions: These results demonstrate for the first time that adiponectin inhibits TNF&agr;-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.