LC3-associated phagocytosis of neutrophils triggers tumor ferroptotic cell death in glioblastoma.

Necrosis in solid tumors is commonly associated with poor prognostic but how these lesions expand remains unclear. Studies have found that neutrophils associate with and contribute to necrosis development in glioblastoma by inducing tumor cell ferroptosis through transferring myeloperoxidase-containing granules. However, the mechanism of neutrophilic granule transfer remains elusive. We performed an unbiased small molecule screen and found that statins inhibit neutrophil-induced tumor cell death by blocking the neutrophilic granule transfer. Further, we identified a novel process wherein neutrophils are engulfed by tumor cells before releasing myeloperoxidase-containing contents into tumor cells. This neutrophil engulfment is initiated by integrin-mediated adhesion, and further mediated by LC3-associated phagocytosis (LAP), which can be blocked by inhibiting the Vps34-UVRAG-RUBCN-containing PI3K complex. Myeloperoxidase inhibition or Vps34 depletion resulted in reduced necrosis formation and prolonged mouse survival in an orthotopic glioblastoma mouse model. Thus, our study unveils a critical role for LAP-mediated neutrophil internalization in facilitating the transfer of neutrophilic granules, which in turn triggers tumor cell death and necrosis expansion. Targeting this process holds promise for improving glioblastoma prognosis.

Iron inhibits glioblastoma cell migration and polarization.

Glioblastoma is one of the deadliest malignancies facing modern oncology today. The ability of glioblastoma cells to diffusely spread into neighboring healthy brain makes complete surgical resection nearly impossible and contributes to the recurrent disease faced by most patients. Although research into the impact of iron on glioblastoma has addressed proliferation, there has been little investigation into how cellular iron impacts the ability of glioblastoma cells to migrate-a key question, especially in the context of the diffuse spread observed in these tumors. Herein, we show that increasing cellular iron content results in decreased migratory capacity of human glioblastoma cells. The decrease in migratory capacity was accompanied by a decrease in cellular polarization in the direction of movement. Expression of CDC42, a Rho GTPase that is essential for both cellular migration and establishment of polarity in the direction of cell movement, was reduced upon iron treatment. We then analyzed a single-cell RNA-seq dataset of human glioblastoma samples and found that cells at the tumor periphery had a gene signature that is consistent with having lower levels of cellular iron. Altogether, our results suggest that cellular iron content is impacting glioblastoma cell migratory capacity and that cells with higher iron levels exhibit reduced motility.

Direct Observation of Compartment-Specific Localization and Dynamics of Voltage-Gated Sodium Channels.

Brain enriched voltage-gated sodium channel (VGSC) Nav1.2 and Nav1.6 are critical for electrical signaling in the central nervous system. Previous studies have extensively characterized cell-type specific expression and electrophysiological properties of these two VGSCs and how their differences contribute to fine-tuning of neuronal excitability. However, due to lack of reliable labeling and imaging methods, the sub-cellular localization and dynamics of these homologous Nav1.2 and Nav1.6 channels remain understudied. To overcome this challenge, we combined genome editing, super-resolution and live-cell single molecule imaging to probe subcellular composition, relative abundances and trafficking dynamics of Nav1.2 and Nav1.6 in cultured mouse and rat neurons and in male and female mouse brain. We discovered a previously uncharacterized trafficking pathway that targets Nav1.2 to the distal axon of unmyelinated neurons. This pathway utilizes distinct signals residing in the intracellular loop 1 (ICL1) between transmembrane domain I and II to suppress the retention of Nav1.2 in the axon initial segment (AIS) and facilitate its membrane loading at the distal axon. As mouse pyramidal neurons undergo myelination, Nav1.2 is gradually excluded from the distal axon as Nav1.6 becomes the dominant VGSC in the axon initial segment and nodes of Ranvier. In addition, we revealed exquisite developmental regulation of Nav1.2 and Nav1.6 localizations in the axon initial segment and dendrites, clarifying the molecular identity of sodium channels in these subcellular compartments. Together, these results unveiled compartment-specific localizations and trafficking mechanisms for VGSCs, which could be regulated separately to modulate membrane excitability in the brain.SIGNIFICANCE STATEMENTDirect observation of endogenous voltage-gated sodium channels reveals a previously uncharacterized distal axon targeting mechanism and the molecular identity of sodium channels in distinct subcellular compartments.

Cepharanthine Alleviates DSS-Induced Ulcerative Colitis via Regulating Aconitate Decarboxylase 1 Expression and Macrophage Infiltration.

Cepharanthine (CEP), a bisbenzylisoquinoline alkaloid from tubers of Stephania, protects against some inflammatory diseases. Aconitate decarboxylase 1 (ACOD1) is also known as immune-responsive gene 1 (IRG1), which plays an important immunometabolism role in inflammatory diseases by mediating the production of itaconic acid. ACOD1 exhibits abnormal expression in ulcerative colitis (UC). However, whether CEP can combat UC by affecting ACOD1 expression remains unanswered. This study was designed to explore the protective effects and mechanisms of CEP in treating colitis through in vitro and in vivo experiments. In vitro assays indicated that CEP inhibited LPS-induced secretion of pro-inflammatory cytokines and ACOD1 expression in RAW264.7 macrophages. Additionally, in the mouse model of DSS-induced colitis, CEP decreased macrophage infiltration and ACOD1 expression in colon tissue. After treatment with antibiotics (Abx), the expression of ACOD1 changed with the composition of gut microbiota. Correlation analysis also revealed that Family-XIII-AD3011-group and Rumini-clostridium-6 were positively correlated with ACOD1 expression level. Additionally, data of the integrative Human Microbiome Project (iHMP) showed that ACOD1 was highly expressed in the colon tissue of UC patients and this expression was positively correlated with the severity of intestinal inflammation. Collectively, CEP can counter UC by modulating gut microbiota and inhibiting the expression of ACOD1. CEP may serve as a potential pharmaceutical candidate in the treatment of UC.

Glucocorticoids enhance the antileukemic activity of FLT3 inhibitors in FLT3-mutant acute myeloid leukemia.

FLT3 is a frequently mutated gene that is highly associated with a poor prognosis in acute myeloid leukemia (AML). Despite initially responding to FLT3 inhibitors, most patients eventually relapse with drug resistance. The mechanism by which resistance arises and the initial response to drug treatment that promotes cell survival is unknown. Recent studies show that a transiently maintained subpopulation of drug-sensitive cells, so-called drug-tolerant "persisters" (DTPs), can survive cytotoxic drug exposure despite lacking resistance-conferring mutations. Using RNA sequencing and drug screening, we find that treatment of FLT3 internal tandem duplication AML cells with quizartinib, a selective FLT3 inhibitor, upregulates inflammatory genes in DTPs and thereby confers susceptibility to anti-inflammatory glucocorticoids (GCs). Mechanistically, the combination of FLT3 inhibitors and GCs enhances cell death of FLT3 mutant, but not wild-type, cells through GC-receptor-dependent upregulation of the proapoptotic protein BIM and proteasomal degradation of the antiapoptotic protein MCL-1. Moreover, the enhanced antileukemic activity by quizartinib and dexamethasone combination has been validated using primary AML patient samples and xenograft mouse models. Collectively, our study indicates that the combination of FLT3 inhibitors and GCs has the potential to eliminate DTPs and therefore prevent minimal residual disease, mutational drug resistance, and relapse in FLT3-mutant AML.

LncRNA XIST facilitates S1P-mediated osteoclast differentiation via interacting with FUS.

INTRODUCTION: The diagnosis and treatment of osteoporosis, a frequent age-related metabolic bone disorder, remain incomprehensive and challenging. The potential regulatory role of lncRNA XIST and sphingosine kinase 1 (SPHK1) pathway need experimental investigations. MATERIALS AND METHODS: RAW264.7 cells and BMMs were obtained for in vitro studies and 30 ng/mL RANKL was implemented for induction of osteoclast differentiation. The suppressing of lncRNA XIST, SPHK1 and fused in sarcoma (FUS) was achieved using small hairpin RNA, while overexpression of XIST and FUS was constructed by pcDNA3.1 vector system. Tartrate-resistant acid phosphatase (TRAP) staining was used for observation of formation of osteoclasts. RNA-pulldown analysis and RNA binding protein immunoprecipitation (RIP) was implemented for measuring mRNA and protein interactions. RT-qPCR was conducted to determining mRNA expression, whereas ELISA and Western blotting assay was performed for monitoring protein expression. RESULTS: RANKL induced osteoclast differentiation and upregulated expression of osteoclastogenesis-related genes that included NFATc1, CTSK, TRAP and SPHK1 and the level of lncRNA XIST in both RAW264.7 cells and BMMs. However, knockdown of lncRNA XIST or suppressing SPHK1 significantly reserved the effects of RANKL. LncRNA XIST was further demonstrated to be interacted with FUS and increased the stability of SPHK1, indicating its ability in promoting osteoclast differentiation through SPHK1/S1P/ERK signaling pathway. CONCLUSION: LncRNA XIST promoted osteoclast differentiation via interacting with FUS and upregulating SPHK1/S1P/ERK pathway.

Time-resolved FRET and NMR analyses reveal selective binding of peptides containing the LC3-interacting region to ATG8 family proteins.

Selective autophagy sequesters cytoplasmic cargo for lysosomal degradation via the binding of autophagy receptors to Atg8 (autophagy-related 8) family proteins on the autophagic membrane. The sole yeast Atg8 gene has six mAtg8 (mammalian Atg8) homologs, including the MAP1LC3 (microtubule-associated protein-1 light chain 3) family and the GABA receptor-associated proteins. Selective autophagy receptors interact with two conserved hydrophobic pockets (termed the W-site and L-site) of mATG8 proteins through a linear motif called the LC3-interacting region (LIR) with the general composition (W/F/Y)XX(I/L/V). To address a lack in our knowledge regarding LIR peptide specificity toward each mATG8 homolog, here we used competitive time-resolved FRET to sensitively and quantitatively characterize the interactions between LIRs and mAtg8. We report that 14 representative LIR-containing peptides display differential binding affinities toward the mAtg8 proteins and identified the LIR domain peptide of TP53INP1 as exhibiting high affinity for all six mATG8 proteins. Using peptide truncation studies, we found that both N- and C-terminal acidic residues, as well as the C-terminal Cys residue of the TP53INP1 LIR peptide, are required for its high-affinity binding to LC3A and LC3B, whereas binding to the GABARAP subfamily proteins was facilitated by residues either N-terminal or C-terminal to the core motif. Finally, we used NMR chemical shift perturbation analysis to gain molecular insights into these findings. Collectively, our results may aid in the development of molecules that selectively disrupt specific mATG8-LIR interactions to dissect the biological roles of the six mATG8 homologs for potential therapeutic applications.

Genistein inhibits amyloid peptide 25-35-induced neuronal death by modulating estrogen receptors, choline acetyltransferase and glutamate receptors.

PURPOSE: To explore genistein, the most active component of soy isoflavones, on viability, expression of estrogen receptor (ER) subtypes, choline acetyltransferase (ChAT), and glutamate receptor subunits in amyloid peptide 25-35-induced hippocampal neurons, providing valuable data and basic information for neuroprotective effect of genistein in Aß25-35-induced neuronal injury. METHODS: We established an in vitro model of Alzheimer's disease by exposing primary hippocampal neurons of newborn rats to amyloid peptide 25-35 (20 µM) for 24 h and observing the effects of genistein (10 µM, 3 h) on viability, expression of ER subtypes, ChAT, NMDA receptor subunit NR2B and AMPA receptor subunit GluR2 in Aß25-35-induced hippocampal neurons. RESULTS: We found that amyloid peptide 25-35 exposure reduced the viability of hippocampal neurons. Meanwhile, amyloid peptide 25-35 exposure decreased the expression of ER subtypes, ChAT and GluR2, and increased the expression of NR2B. Genistein at least partially reversed the effects of amyloid peptide 25-35 in hippocampal neurons. CONCLUSION: Genistein could increase the expression of ChAT as a consequence of activating estrogen receptor subtypes, modulating the expression of NR2B and GluR2, and thereby ameliorating the status of hippocampal neurons and exerting neuroprotective effects against amyloid peptide 25-35. Our data suggest that genistein might represent a potential cell-targeted therapy which could be a promising approach to treating AD.

Pivotal role of mitophagy in response of acute myelogenous leukemia to a ceramide-tamoxifen-containing drug regimen.

Acute myelogenous leukemia (AML) is a hematological malignancy marked by the accumulation of large numbers of immature myeloblasts in bone marrow. The overall prognosis in AML is poor; hence, there is a pressing need to improve treatment. Although the sphingolipid (SL) ceramide demonstrates known cancer suppressor properties, it's mechanism of action is multifaceted. Our studies in leukemia and other cancers have demonstrated that when combined with the antiestrogen, tamoxifen, the apoptosis-inducting effect of ceramide is greatly enhanced. The goal of the present study was to establish whether a ceramide-tamoxifen regimen also affects autophagic-driven cellular responses in leukemia. Using the human AML cell line KG-1, we demonstrate that, unlike exposure to the single agents, combination C6-ceramide-tamoxifen upregulated LC3-II expression, inhibited the mTOR signaling pathway, and synergistically induced KG-1 cell death in an Atg5-dependent manner. In addition, colocalization of autophagosome and mitochondria, indicative of mitophagosome formation and mitophagy, was observed. Versatility of the drug regimen was confirmed by experiments in MV4-11 cells, a FLT3-ITD AML mutant. These results indicate that the C6-ceramide-tamoxifen regimen plays a pivotal role inducing autophagy in AML, and thus constitutes a novel therapeutic design.

Loss of E2F7 confers resistance to poly-ADP-ribose polymerase (PARP) inhibitors in BRCA2-deficient cells.

BRCA proteins are essential for homologous recombination (HR) DNA repair, and their germline or somatic inactivation is frequently observed in human tumors. Understanding the molecular mechanisms underlying the response of BRCA-deficient tumors to chemotherapy is paramount for developing improved personalized cancer therapies. While PARP inhibitors have been recently approved for treatment of BRCA-mutant breast and ovarian cancers, not all patients respond to this therapy, and resistance to these novel drugs remains a major clinical problem. Several mechanisms of chemoresistance in BRCA2-deficient cells have been identified. Rather than restoring normal recombination, these mechanisms result in stabilization of stalled replication forks, which can be subjected to degradation in BRCA2-mutated cells. Here, we show that the transcriptional repressor E2F7 modulates the chemosensitivity of BRCA2-deficient cells. We found that BRCA2-deficient cells are less sensitive to PARP inhibitor and cisplatin treatment after E2F7 depletion. Moreover, we show that the mechanism underlying this activity involves increased expression of RAD51, a target for E2F7-mediated transcriptional repression, which enhances both HR DNA repair, and replication fork stability in BRCA2-deficient cells. Our work describes a new mechanism of therapy resistance in BRCA2-deficient cells, and identifies E2F7 as a putative biomarker for tumor response to PARP inhibitor therapy.

Bif-1 Interacts with Prohibitin-2 to Regulate Mitochondrial Inner Membrane during Cell Stress and Apoptosis.

BACKGROUND: Mitochondria are dynamic organelles that undergo fission and fusion. During cell stress, mitochondrial dynamics shift to fission, leading to mitochondrial fragmentation, membrane leakage, and apoptosis. Mitochondrial fragmentation requires the cleavage of both outer and inner membranes, but the mechanism of inner membrane cleavage is unclear. Bif-1 and prohibitin-2 may regulate mitochondrial dynamics. METHODS: We used azide-induced ATP depletion to incite cell stress in mouse embryonic fibroblasts and renal proximal tubular cells, and renal ischemia-reperfusion to induce stress in mice. We also used knockout cells and mice to determine the role of Bif-1, and used multiple techniques to analyze the molecular interaction between Bif-1 and prohibitin-2. RESULTS: Upon cell stress, Bif-1 translocated to mitochondria to bind prohibitin-2, resulting in the disruption of prohibitin complex and proteolytic inactivation of the inner membrane fusion protein OPA1. Bif-1-deficiency inhibited prohibitin complex disruption, OPA1 proteolysis, mitochondrial fragmentation, and apoptosis. Domain deletion analysis indicated that Bif-1 interacted with prohibitin-2 via its C-terminus. Notably, mutation of Bif-1 at its C-terminal tryptophan-344 not only prevented Bif-1/prohibitin-2 interaction but also reduced prohibitin complex disruption, OPA1 proteolysis, mitochondrial fragmentation, and apoptosis, supporting a pathogenic role of Bif-1/prohibitin-2 interaction. In mice, Bif-1 bound prohibitin-2 during renal ischemia/reperfusion injury, and Bif-1-deficiency protected against OPA1 proteolysis, mitochondrial fragmentation, apoptosis and kidney injury. CONCLUSIONS: These findings suggest that during cell stress, Bif-1 regulates mitochondrial inner membrane by interacting with prohibitin-2 to disrupt prohibitin complexes and induce OPA1 proteolysis and inactivation.

Acid ceramidase promotes drug resistance in acute myeloid leukemia through NF-κB-dependent P-glycoprotein upregulation.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. More than half of older AML patients fail to respond to cytotoxic chemotherapy, and most responders relapse with drug-resistant disease. Failure to achieve complete remission can be partly attributed to the drug resistance advantage of AML blasts that frequently express P-glycoprotein (P-gp), an ATP-binding cassette transporter. Our previous work showed that elevated acid ceramidase (AC) levels in AML contribute to blast survival. Here, we investigated P-gp expression levels in AML relative to AC. Using parental HL-60 cells and drug-resistant derivatives as our model, we found that P-gp expression and efflux activity were highly upregulated in resistant derivatives. AC overexpression in HL-60 conferred resistance to the AML chemotherapeutic drugs, cytarabine, mitoxantrone, and daunorubicin, and was linked to P-gp upregulation. Furthermore, targeting AC through pharmacologic or genetic approaches decreased P-gp levels and increased sensitivity to chemotherapeutic drugs. Mechanistically, AC overexpression increased NF-κB activation whereas NF-kB inhibitors reduced P-gp levels, indicating that the NF-kappaB pathway contributes to AC-mediated modulation of P-gp expression. Hence, our data support an important role for AC in drug resistance as well as survival and suggest that sphingolipid targeting approaches may also impact drug resistance in AML.

Postnatal Expression Patterns of Estrogen Receptor Subtypes and Choline Acetyltransferase in Different Regions of the Papez Circuit.

The Papez circuit is crucial for several brain functions, including long-term memory and emotion. Estradiol modulates cognitive functions based on the expression pattern of its receptor subtypes including estrogen receptor (ER) α, ß, and G protein-coupled receptor 30 (GPR30). Similarly, the activity in the cholinergic system correlates with several brain functions, such as learning and memory. In this study, we used immunofluorescence to examine the expression patterns of ERß and Western blotting to analyze GPR30 and choline acetyltransferase (ChAT) expression, in different regions of the Papez circuit, including the prefrontal cortex, hippocampus, hypothalamus, anterior nucleus of the thalamus, and cingulum in female rats at postnatal days (PND) 1, 10, and 56. Our main finding was that the highest expression of ERß and GPR30 was noted in each brain area of the Papez circuit in the PND1 rats, whereas the expression of ChAT was the highest in PND10 rats. These results provide vital information on the postnatal expression patterns of ER subtypes and ChAT in different regions of the Papez circuit.

Genistein Inhibits Aß25-35-Induced Neuronal Death with Changes in the Electrophysiological Properties of Voltage-Gated Sodium and Potassium Channels.

We established a model of Alzheimer's disease in vitro by exposing primary hippocampal neurons of neonatal Wistar rats to the ß-Amyloid peptide fragment 25-35, Aß25-35. We then observed the effects of genistein, a type of soybean isoflavone, on Aß25-35-incubated hippocampal neuron viability, and the electrophysiological properties of voltage-gated sodium channels (NaV) and potassium channels (KV) in the hippocampal neurons. Aß25-35 exposure reduced the viability of hippocampal neurons, decreased the peak amplitude of voltage-activated sodium channel currents (INa), and significantly reduced INa at different membrane potentials. Moreover, Aß25-35 shifted the activation curve toward depolarization, shifted the inactivation curve toward hyperpolarization, and increased the time constant of recovery from inactivation. Aß25-35 exposure significantly shifted the inactivation curve of transient outward K+ currents (IA) toward hyperpolarization and increased its time constant of recovery from inactivation. In addition, Aß25-35 significantly decreased the peak density of outward-delayed rectifier potassium channel currents (IDR) and significantly reduced IDR value at different membrane potentials. We found that genistein partially reversed the decrease in hippocampal neuron viability, and the alterations in electrophysiological properties of NaV and KV induced by Aß25-35. Our results suggest that genistein could inhibit Aß25-35-induced neuronal damage with changes in the electrophysiological properties of NaV and KV.

Genistein inhibits Aß25-35 -induced SH-SY5Y cell damage by modulating the expression of apoptosis-related proteins and Ca2+ influx through ionotropic glutamate receptors.

In this study, we investigated the protective effects of genistein against SH-SY5Y cell damage induced by ß-amyloid 25-35 peptide (Aß25-35 ) and the underlying mechanisms. Aß-induced neuronal death, apoptosis, glutamate receptor subunit expression, Ca2+ ion concentration, amino acid transmitter concentration, and apoptosis-related factor expression were evaluated to determine the effects of genistein on Aß-induced neuronal death and apoptosis. The results showed that genistein increased the survival of SH-SY5Y cells and decreased the level of apoptosis induced by Aß25-35 . In addition, genistein reversed the Aß25-35 -induced changes in amino acid transmitters, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and N-methyl-d-aspartate (NMDA) receptor subunits in SH-SY5Y cells. Aß25-35 -induced changes in Ca2+ and B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X (Bax) protein and gene levels in cells were also reversed by genistein. Our data suggest that genistein protects against Aß25-35 -induced damage in SH-SY5Y cells, possibly by regulating the expression of apoptosis-related proteins and Ca2+ influx through ionotropic glutamate receptors.

Endophilin B2 facilitates endosome maturation in response to growth factor stimulation, autophagy induction, and influenza A virus infection.

Endocytosis, and the subsequent trafficking of endosomes, requires dynamic physical alterations in membrane shape that are mediated in part by endophilin proteins. The endophilin B family of proteins contains an N-terminal Bin/amphiphysin/Rvs (N-BAR) domain that induces membrane curvature to regulate intracellular membrane dynamics. Whereas endophilin B1 (SH3GLB1/Bif-1) is known to be involved in a number of cellular processes, including apoptosis, autophagy, and endocytosis, the cellular function of endophilin B2 (SH3GLB2) is not well understood. In this study, we used genetic approaches that revealed that endophilin B2 is not required for embryonic development in vivo but that endophilin B2 deficiency impairs endosomal trafficking in vitro, as evidenced by suppressed endosome acidification, EGFR degradation, autophagic flux, and influenza A viral RNA nuclear entry and replication. Mechanistically, although the loss of endophilin B2 did not affect endocytic internalization and lysosomal function, endophilin B2 appeared to regulate the trafficking of endocytic vesicles and autophagosomes to late endosomes or lysosomes. Moreover, we also found that despite having an intracellular localization and tissue distribution similar to endophilin B1, endophilin B2 is dispensable for mitochondrial apoptosis. Taken together, our findings suggest that endophilin B2 positively regulates the endocytic pathway in response to growth factor signaling, autophagy induction, and viral entry.

Depletion of the Human Ion Channel TRPM2 in Neuroblastoma Demonstrates Its Key Role in Cell Survival through Modulation of Mitochondrial Reactive Oxygen Species and Bioenergetics.

Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in modulating cell survival following oxidant injury and is highly expressed in many cancers including neuroblastoma. Here, in xenografts generated from neuroblastoma cells in which TRPM2 was depleted with CRISPR/Cas9 technology and in in vitro experiments, tumor growth was significantly inhibited and doxorubicin sensitivity increased. The hypoxia-inducible transcription factor 1/2α (HIF-1/2α) signaling cascade including proteins involved in oxidant stress, glycolysis, and mitochondrial function was suppressed by TRPM2 depletion. TRPM2-depleted SH-SY5Y neuroblastoma cells demonstrated reduced oxygen consumption and ATP production after doxorubicin, confirming impaired cellular bioenergetics. In cells in which TRPM2 was depleted, mitochondrial superoxide production was significantly increased, particularly following doxorubicin. Ectopic expression of superoxide dismutase 2 (SOD2) reduced ROS and preserved viability of TRPM2-depleted cells, however, failed to restore ATP levels. Mitochondrial reactive oxygen species (ROS) were also significantly increased in cells in which TRPM2 function was inhibited by TRPM2-S, and pretreatment of these cells with the antioxidant MitoTEMPO significantly reduced ROS levels in response to doxorubicin and protected cell viability. Expression of the TRPM2 pore mutant E960D, in which calcium entry through TRPM2 is abolished, also resulted in significantly increased mitochondrial ROS following doxorubicin treatment, showing the critical role of TRPM2-mediated calcium entry. These findings demonstrate the important function of TRPM2 in modulation of cell survival through mitochondrial ROS, and the potential of targeted inhibition of TRPM2 as a therapeutic approach to reduce cellular bioenergetics, tumor growth, and enhance susceptibility to chemotherapeutic agents.

TPEN, a Specific Zn2+ Chelator, Inhibits Sodium Dithionite and Glucose Deprivation (SDGD)-Induced Neuronal Death by Modulating Apoptosis, Glutamate Signaling, and Voltage-Gated K+ and Na+ Channels.

Hypoxia-ischemia-induced neuronal death is an important pathophysiological process that accompanies ischemic stroke and represents a major challenge in preventing ischemic stroke. To elucidate factors related to and a potential preventative mechanism of hypoxia-ischemia-induced neuronal death, primary neurons were exposed to sodium dithionite and glucose deprivation (SDGD) to mimic hypoxic-ischemic conditions. The effects of N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn2+-chelating agent, on SDGD-induced neuronal death, glutamate signaling (including the free glutamate concentration and expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor (GluR2) and N-methyl-D-aspartate (NMDA) receptor subunits (NR2B), and voltage-dependent K+ and Na+ channel currents were also investigated. Our results demonstrated that TPEN significantly suppressed increases in cell death, apoptosis, neuronal glutamate release into the culture medium, NR2B protein expression, and I K as well as decreased GluR2 protein expression and Na+ channel activity in primary cultured neurons exposed to SDGD. These results suggest that TPEN could inhibit SDGD-induced neuronal death by modulating apoptosis, glutamate signaling (via ligand-gated channels such as AMPA and NMDA receptors), and voltage-gated K+ and Na+ channels in neurons. Hence, Zn2+ chelation might be a promising approach for counteracting the neuronal loss caused by transient global ischemia. Moreover, TPEN could represent a potential cell-targeted therapy.

A novel NaV1.5 voltage sensor mutation associated with severe atrial and ventricular arrhythmias.

BACKGROUND: Inherited autosomal dominant mutations in cardiac sodium channels (NaV1.5) cause various arrhythmias, such as long QT syndrome and Brugada syndrome. Although dozens of mutations throughout the protein have been reported, there are few reported mutations within a voltage sensor S4 transmembrane segment and few that are homozygous. Here we report analysis of a novel lidocaine-sensitive recessive mutation, p.R1309H, in the NaV1.5 DIII/S4 voltage sensor in a patient with a complex arrhythmia syndrome. METHODS AND RESULTS: We expressed the wild type or mutant NaV1.5 heterologously for analysis with the patch-clamp and voltage clamp fluorometry (VCF) techniques. p.R1309H depolarized the voltage-dependence of activation, hyperpolarized the voltage-dependence of inactivation, and slowed recovery from inactivation, thereby reducing the channel availability at physiologic membrane potentials. Additionally, p.R1309H increased the "late" Na(+) current. The location of the mutation in DIIIS4 prompted testing for a gating pore current. We observed an inward current at hyperpolarizing voltages that likely exacerbates the loss-of-function defects at resting membrane potentials. Lidocaine reduced the gating pore current. CONCLUSIONS: The p.R1309H homozygous NaV1.5 mutation conferred both gain-of-function and loss-of-function effects on NaV1.5 channel activity. Reduction of a mutation-induced gating pore current by lidocaine suggested a therapeutic mechanism.

Tid1, the Mammalian Homologue of Drosophila Tumor Suppressor Tid56, Mediates Macroautophagy by Interacting with Beclin1-containing Autophagy Protein Complex.

One of the fundamental functions of molecular chaperone proteins is to selectively conjugate cellular proteins, targeting them directly to lysosome. Some of chaperones, such as the stress-induced Hsp70, also play important roles in autophagosome-forming macroautophagy under various stress conditions. However, the role of their co-chaperones in autophagy regulation has not been well defined. We here show that Tid1, a DnaJ co-chaperone for Hsp70 and the mammalian homologue of the Drosophila tumor suppressor Tid56, is a key mediator of macroautophagy pathway. Ectopic expression of Tid1 induces autophagy by forming LC3+ autophagosome foci, whereas silencing Tid1 leads to drastic impairment of autophagy as induced by nutrient deprivation or rapamycin. In contrast, Hsp70 is dispensable for a role in nutrient deprivation-induced autophagy. The murine Tid1 can be replaced with human Tid1 in murine fibroblast cells for induction of autophagy. We further show that Tid1 increases autophagy flux by interacting with the Beclin1-PI3 kinase class III protein complex in response to autophagy inducing signal and that Tid1 is an essential mediator that connects IκB kinases to the Beclin1-containing autophagy protein complex. Together, these results reveal a crucial role of Tid1 as an evolutionarily conserved and essential mediator of canonical macroautophagy.