Biology subjects

Hempel, N.

Publications and source records attributed to Hempel, N..

24 records · Page 2Linked to original sources

bioRxiv2021

Regulation of Interorganellar Ca2+ Transfer and NFAT Activation by the Mitochondrial Ca2+ Uniporter

Mitochondrial Ca2+ uptake is crucial for coupling receptor stimulation to cellular bioenergetics. Further, Ca2+ uptake by respiring mitochondria prevents Ca2+-dependent inactivation (CDI) of store-operated Ca2+ release-activated Ca2+ (CRAC) channels and inhibits Ca2+ extrusion to sustain cytosolic Ca2+ signaling. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we generated several cell lines with MCU-knockout (MCU-KO) as well as tissue-specific MCU-knockdown mice. We show that mitochondrial depolarization, but not MCU-KO, inhibits store-operated Ca2+ entry (SOCE). Paradoxically, despite enhancing Ca2+ extrusion and promoting CRAC channel CDI, MCU-KO increased cytosolic Ca2+ in response to store depletion. Further, physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, NFAT nuclear translocation and proliferation. However, MCU-KO did not affect inositol-1,4,5-trisphosphate receptor activity. Mathematical modeling supports that MCU-KO enhances cytosolic Ca2+, despite limiting CRAC channel activity.

cell biology↗

bioRxiv2021

Arc rescues defective synaptic plasticity and cognitive dysfunction in sepsis-associated encephalopathy

Sepsis-associated encephalopathy (SAE) is a frequent complication in patients with severe systemic infection resulting in acute brain dysfunction and incapacitating long-term sequelae. SAE includes delirium, premature death, post-traumatic stress disorder, and major long-term cognitive impairment. The underlying pathophysiology of SAE is largely unresolved and specific treatment options are missing. We induced the peritoneal contamination and infection (PCI) sepsis model in 769 mice and compared these with 259 control mice. We found that experimental sepsis causes synaptic pathology in the brain characterized by severely disordered synaptic plasticity with reduced long-term potentiation, changes in CA1 pyramidal neuron dendritic spines, and behavioral abnormalities indicating cognitive dysfunction. Using electrophysiology, we found reduced frequency of quantal and spontaneous excitatory postsynaptic currents whereas amplitudes of miniature, spontaneous, and evoked excitatory currents were increased, pointing towards a homeostatic synaptic scaling mechanism. Corresponding to dysfunctional excitatory synaptic function we discovered downregulation of genes related to neuronal and synaptic signaling in the brain, including the gene for activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), members of the transcription-regulatory EGR gene family, and the gene for dual-specificity phosphatase 6 (Dusp6). At the protein level, ARC expression and MAP kinase signaling in the brain were affected. For targeted rescue of dysfunctional synaptic signaling and plasticity, we overexpressed ARC in the hippocampus by microinjection of an adeno-associated virus containing a neuron-specific plasmid of the ARC transgene. Hereby we achieved recovery of defective synaptic plasticity in the hippocampal Schaffer collateral-CA1 pathway and improvement of memory dysfunction. Using a different rescue paradigm, PCI mice were subjected to enriched environment providing multiple activating stimuli. Enriched environment led to restoration of disordered long-term potentiation and memory, thus demonstrating the potential for activity-induced improvement. Together, we identified synaptic pathomechanisms of SAE after severe systemic infection and provide a conceptual approach to treat SAE-related disease mechanisms which may be applicable to patients afflicted with SAE.

neuroscience↗

bioRxiv2021

HuR -dependent SOD2 protein synthesis is an early adaptation of ovarian cancer cells in response to anchorage independence

During metastasis, cancer cells must adapt to survive a loss of anchorage and evade anoikis. An important pro-survival adaptation is the ability of metastatic tumor cells to increase their antioxidant capacity and restore cellular redox balance. Although much is known about the transcriptional regulation of antioxidant enzymes in response to stress, how cells rapidly adapt to alter antioxidant enzyme levels is less well understood. Using ovarian cancer cells as a model, we demonstrate that an increase in protein expression of the mitochondrial superoxide dismutase SOD2 is a very early event initiated in response to cellular detachment. SOD2 protein synthesis is rapidly induced within 0.5-2 hours of matrix detachment, and polyribosome profiling demonstrates an increase in the number of ribosomes bound to SOD2 mRNA, indicating an increase in SOD2 translation in response to anchorage-independence. Mechanistically, we find that anchorage-independence specifically induces cytosolic accumulation of the RNA binding protein HuR/ELAVL1 and leads to increased HuR binding to SOD2 mRNA. Using HuR siRNA-mediated knock-down, we show that the presence of HuR is necessary for the increase in SOD2 mRNA association with the heavy polyribosome fraction and SOD2 protein synthesis observed in anchorage-independence. Cellular detachment activates the stress-response protein kinase p38 MAPK, which is necessary for HuR-SOD2 mRNA binding and optimal increases in SOD2 protein expression. These findings illustrate a novel post-transcriptional regulatory mechanisms of SOD2, enabling cells to rapidly increase their mitochondrial antioxidant capacity as an acute response to anchorage-independence.

cancer biology↗

bioRxiv2020

Omnitemporal choreographies of IP3R and all five STIM/Orai underlie the complexity of mammalian Ca2+ signaling

Invertebrates express one endoplasmic reticulum (ER)-resident Ca2+-sensing stromal-interaction molecule (Stim) and one Orai plasma membrane channel protein. Stim conveys store depletion to Orai, mediating the evolutionarily conserved Ca2+ release-activated Ca2+ (CRAC) current. The crucial role of their vertebrate homologues, STIM1 and Orai1 in mediating CRAC activity in mammals is well-established. However, mammals possess two STIM and three Orai isoforms and the choreography of their interactions under physiological receptor activation is unknown. We show that the five mammalian STIM1/2 and Orai1/2/3 isoforms have non-redundant functions. Yet, all five isoforms are always required together to ensure the graded diversity of mammalian Ca2+ signaling events in response to the full spectrum of agonist strengths. Receptor-activated Ca2+ signaling across the range of stimulus intensities requires functional interactions between not only STIM1/2 and Orai1/2/3, but also IP3R, ensuring that receptor-mediated Ca2+ release is precisely tailored to Ca2+ entry and activation of nuclear factor of activated T-cells (NFAT). This is orchestrated by two interdependent and counterbalancing paradigms: the N-termini Ca2+-binding ER-luminal domains of unactivated STIM1/2 inhibit IP3R-evoked Ca2+ release. Gradual increase in agonist intensity leads to gradual STIM1/2 activation and relief of IP3R inhibition. Concomitantly, the cytosolic C-termini of activated STIM1/2 differentially interact with Orai1/2/3 proteins as agonist intensity increases. Thus, coordinated and omnitemporal functions of all five STIM/Orai proteins and IP3Rs at the ER-lumen and cytosol translate the strength of agonist stimulation to precise levels of Ca2+ release, Ca2+ entry and NFAT induction, ensuring the diversity and fidelity of complex mammalian Ca2+ signaling. HighlightsO_LIAll five STIM/Orai and IP3R are always required together in mammalian Ca2+ signalling C_LIO_LIUnactivated STIM1/2 inhibit IP3R and activated STIM1/2 cooperatively activate Orai1/2/3 C_LIO_LISTIM1 contribution increases and that of STIM2 decreases as agonist intensifies C_LIO_LIGraded IP3R disinhibition and Orai activation tailor receptor activity to NFAT induction C_LI O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/325480v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@f6172dorg.highwire.dtl.DTLVardef@6bbb09org.highwire.dtl.DTLVardef@c45c80org.highwire.dtl.DTLVardef@194d7e4_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2020

Colorectal adenocarcinomas downregulate the mitochondrial Na+/Ca2+ exchanger NCLX to drive metastatic spread

Despite the established role of mitochondria in tumorigenesis, the molecular mechanisms by which mitochondrial Ca2+ (mtCa2+) signaling regulates tumor growth and metastasis remain unknown. The crucial role of mtCa2+ in tumorigenesis is highlighted by the altered expression of proteins mediating mtCa2+ uptake and extrusion in cancer cells. Here, we demonstrate that expression of the mitochondrial Na+/Ca2+ exchanger NCLX (SLC8B1) is decreased in colorectal tumors and is associated with advanced-stage disease in patients. We reveal that downregulation of NCLX leads to mtCa2+ overload, mitochondrial depolarization, mitophagy, and reduced tumor size. Concomitantly, NCLX downregulation drives metastatic spread, chemoresistance, the expression of epithelial-to-mesenchymal transition (EMT), hypoxia, and stem cell pathways. Mechanistically, mtCa2+ overload leads to an increase in mitochondrial reactive oxygen species (mtROS) which activates HIF1 signaling supporting the metastatic behavior of tumor cells lacking NCLX. Our results reveal that loss of NCLX expression is a novel driver of metastatic progression, indicating that control of mtCa2+ levels is a novel therapeutic approach in metastatic colorectal cancer. HighlightsO_LIThe expression of NCLX is decreased in colorectal tumors and is associated with advanced-stage disease in patients. C_LIO_LINCLX plays a dichotomous role in colorectal tumor growth and metastasis. C_LIO_LINCLX downregulation causes mitophagy and reduced colorectal cancer tumor growth. C_LIO_LINCLX downregulation induces stemness, chemoresistance and metastasis through mtCa2+/ROS/HIF1 signaling axis. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/083071v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@30360dorg.highwire.dtl.DTLVardef@12ca72borg.highwire.dtl.DTLVardef@9328ddorg.highwire.dtl.DTLVardef@15ffcdb_HPS_FORMAT_FIGEXP M_FIG C_FIG SignificanceMitochondrial Ca2+ (mtCa2+) homeostasis is essential for cellular metabolism and growth and plays a critical role in cancer progression. mtCa2+ uptake is mediated by an inner membrane protein complex containing the mitochondrial Ca2+ uniporter (MCU). mtCa2+ uptake by the MCU is followed by a [~]100-fold slower mtCa2+ extrusion mediated by the inner mitochondrial membrane ion transporter, the mitochondrial Na+/Ca2+ exchanger NCLX. Because NCLX is a slower transporter than the MCU, it is a crucial rate-limiting factor of mtCa2+ homeostasis that cannot easily be compensated by another Ca2+ transport mechanism. This represents the first study investigating the role of NCLX in tumorigenesis and metastasis. We demonstrate for the first time that colorectal cancers exhibit loss of NCLX expression and that this is associated with advanced-stage disease. Intriguingly, decreased NCLX function has a dichotomous role in colorectal cancer. Thus, we reveal that NCLX loss leads to reduced primary tumor growth and overall tumor burden in vivo. Yet, the consequential increases in mtCa2+ elicit pro-survival, hypoxic and gene transcription pathways that enhance metastatic progression. This dichotomy is a well-established feature of chemoresistant and recurrent tumor cells including cancer stem cells. Moreover, the downstream changes elicited by NCLX loss are reminiscent of mesenchymal colorectal cancer subtypes that display poor patient survival. Our data indicate that the demonstrated changes to the mtCa2+/mtROS/HIF1 signaling axis elicited through the loss of NCLX are a key adaptation and driver of metastatic colorectal cancer.

cancer biology↗

bioRxiv2019

Context-dependent activation of SIRT3 is necessary for anchorage-independent survival and metastasis of ovarian cancer cells.

Cells must alter their antioxidant capacity for maximal metastatic potential. However, the antioxidant adaptations required for transcoelomic metastasis, which is the passive dissemination of cancer cells in the peritoneal cavity as seen in ovarian cancer, have largely remained unexplored. Contradicting the need for oxidant scavenging by tumor cells is the observation that expression of the nutrient stress sensor and regulator of mitochondrial antioxidant defenses, SIRT3, is suppressed in many primary tumors. We discovered that this mitochondrial deacetylase is however, upregulated in a context-dependent manner in cancer cells. SIRT3 activity and expression transiently increased following ovarian cancer cell detachment and in tumor cells derived from malignant ascites of high-grade serous adenocarcinoma patients. Mechanistically, SIRT3 prevents mitochondrial superoxide surges in detached cells by regulating the manganese superoxide dismutase SOD2. This mitochondrial stress response is under dual regulation by SIRT3. SIRT3 rapidly increases SOD2 activity as an early adaptation to cellular detachment, which is followed by SIRT3-dependent transcriptional increases in SOD2 during sustained anchorage-independence. In addition, SIRT3 inhibits glycolytic capacity in anchorage-independent cells thereby contributing to metabolic changes in response to detachment. While manipulation of SIRT3 expression has few deleterious effects on cancer cells in attached conditions, SIRT3 up-regulation and SIRT3-mediated oxidant scavenging following matrix detachment are required for anoikis resistance in vitro, and both SIRT3 and SOD2 are necessary for colonization of the peritoneal cavity in vivo. Our results highlight the novel context-specific, pro-metastatic role of SIRT3 in ovarian cancer.

cancer biology↗