Bisindolylmaleimide Research Articles

Filamin A regulates platelet shape change and contractile force generation via phosphorylation of the myosin light chain.

Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin cross-linking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner.

Activation of PKC affects the ventricular restitution properties and arrhythmogenesis through L-type Ca+ current.

To investigate the role of protein kinase C (PKC) in action potential duration (APD) restitution and ventricular tachyarrhythmias (VAs). Rabbits hearts were isolated and prepared for Langendorff perfusion technique. The stimuli-extra-stimulus (S1-S2) method and dynamic S1 pacing protocol were performed to construct APD restitution and to induce APD alternans or VA, respectively, at 10 sites throughout the ventricular chamber. Administration of phorbol-12-myristate-13-acetate (PMA) (100nM) (n=15) greatly steepened the restitution curves (Smax>1) (p<.01) at each site compared to the control group (n=15). Furthermore, treatment with PMA also induced larger spatial dispersions of Smax (p<.05) and decreased the thresholds of the VA and APD alternans (p<.01). However, perfused with the PKC inhibitor, bisindolylmaleimide (BIM) (500nM) (n=10), reversibly flattened the APD restitution curves at each site (Smax<1), decreased the spatial dispersions of Smax, and increased the thresholds of APD alternans and VA. According to the results of patch-clamp, peak amplitude of L-type Ca2+ current was significantly increased by addition of PMA compared with control (CTL) group (p<.05). Antagonize this current with verapamil (n=10) can fully inhibited the PMA induced increasing of Smax and inducibility of VA and alternans. PKC activation increased the dispersion of APD restitution and thus led to occurrence of VA, which possibly related to the increased Ca2+ influx.

Bisindolyl Maleimides and Indolylmaleimide Derivatives-A Review of Their Synthesis and Bioactivity.

The evolution of bisindolyl maleimides and indolyl maleimide derivatives and their unique biological activities have stimulated great interest in medicinal chemistry programs. Bisindolylmaleimide (BIM)-type compounds arise from natural sources such as arcyriarubin and are biosynthetically related to indolocarbazoles. BIMs are commonly the immediate synthetic precursors of indolocarbazoles, lacking a central bond between the two aromatic units and making them more flexible and drug-like. Synthetic endeavours within this class of compounds are broad and have led to the development of both remarkably potent and selective protein kinase inhibitors. Clinical BIM examples include ruboxistaurin and enzastaurin, which are highly active inhibitors of protein kinase C-β. While BIMs are widely recognised as protein kinase inhibitors, other modes of activity have been reported, including the inhibition of calcium signalling and antimicrobial activity. Critically, structural differences can be used to exploit new bioactivity and therefore it is imperative to discover new chemical entities to address new targets. BIMs can be highly functionalised or chemically manipulated, which provides the opportunity to generate new derivatives with unique biological profiles. This review will collate new synthetic approaches to BIM-type compounds and their associated bioactivities with a focus on clinical applications.

Metabolic regulation of the two-pore domain potassium channel TASK-1

TASK-1 is a two-pore potassium ion channel and implicated in the maintenance of cell membrane potential. Little is known about the traffic regulation of TASK-1 under metabolic stress. The present study was designed to explore whether cell surface expression of TASK-1 is modulated by hypoxia. The trafficking of recombinant TASK-1 channels was studied in HEK293T cells under hypoxia induced by sodium cyanide (NaCN). Incubation of cells with NaCN significantly reduced TASK-1 fluorescence intensity at cell periphery, and this effect was inhibited by pretreatment with bisindolylmaleimide (BIM), a specific protein kinase C (PKC) inhibitor. Similar results were confirmed by quantifying extracellular HA-tagged TASK-1 on the cell surface using chemiluminescence assay. NaCN-induced reduction in TASK-1 at cell periphery was also prevented by co-expressing the dominant-negative dynamin 1 (K44E), or cholesterol depletion with MβCD, indicating a potential role of cholesterol and dynamin-dependent endocytosis in PKC-mediated reduction of cell surface TASK-1 by NaCN. Mutagenesis studies further showed that NaCN-induced reduction of TASK-1 at cell periphery was observed in cells expressing wild-type TASK-1 but not mutant TASK-1 with mutation of the dileucine motif (263-264 LL-AA) or the PKC phosphorylation consensus site (T400A). Moreover, Western blot analysis demonstrated that NaCN reduced the amount of wild-type TASK-1 but not the TASK-1 mutants (263-264 LL-AA and T400A) in the membrane fraction. In conclusion, our results demonstrate that cell surface TASK-1 channels are downregulated under metabolic inhibition, possibly through PKC-mediated endocytosis in a cholesterol and dynamin-dependent manner. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Filamin A regulates caspase-3 cleavage in platelets in a protein kinase C (PKC)-dependent manner.

Apoptosis is a critical process for the maintenance of cell populations, and involves mitochondrial depolarization, the sequential cleavage of caspase-9 and -3, followed by the externalization of phosphatidylserine (PS) on the plasma membrane. The actin cytoskeleton and its accessory proteins are known regulators of apoptotic signaling in nucleated cells but their roles in platelet apoptosis are undefined. Filamin A (FLNA) is a ubiquitously expressed actin-crosslinking protein that also serves as an intracellular signaling scaffold. Here we used platelets from mice with a platelet-specific FLNA deficiency (Flnafl/Y, Pf4-cre/+, termed platelet-specific knockout) to test the role of FLNA in platelet apoptosis. Treatment with the BH3-mimetic drug ABT-737 induced caspase-3 cleavage and PS exposure in platelets from floxed mice (Flnafl/Y, termed control) but these effects were essentially abrogated in FLNA-null platelets (platelet-specific knockout). Protein kinase C (PKC), a known FLNA ligand, was also activated by ABT-737, and PKC's phosphorylation of its downstream substrates was attenuated in FLNA-null platelets. The PKC inhibitor bisindolylmaleimide (BIM) also reduced caspase-3 cleavage, thus essentially phenocopying the FLNA-null platelets. Notably, the caspase-3 cleavage defect in FLNA-null platelets was rescued by the PKC-activating phorbol ester PMA, suggesting that FLNA and PKC share a common pathway in regulating platelet apoptosis. Mitochondrial depolarization and caspase-9 cleavage were unaffected by BIM treatment, suggesting that PKC specifically controls the downstream caspase-3 point of the pro-apoptotic signaling pathway. These data point to a novel role for FLNA in the regulation of platelet apoptosis, thus providing an improved understanding of how circulating platelet counts are maintained.

Crosstalk between protein kinases A and C regulates sea urchin sperm motility.

Fertilization, a crucial event for species preservation, in sea urchins, as in many other organisms, requires sperm motility regulation. In Strongylocentrotus purpuratus sea urchins, speract, a sperm chemoattractant component released to seawater from the outer egg layer, attracts sperm after binding to its receptor in the sperm flagellum. Previous experiments performed in demembranated sperm indicated that motility regulation in these cells involved protein phosphorylation mainly due to the cAMP-dependent protein kinase (PKA). However, little information is known about the involvement of protein kinase C (PKC) in this process. In this work, using intact S. purpuratus sea urchin sperm, we show that: (i) the levels of both phosphorylated PKA (PKA substrates) and PKC (PKC substrates) substrates change between immotile, motile and speract-stimulated sperm, and (ii) the non-competitive PKA (H89) and PKC (chelerythrine) inhibitors diminish the circular velocity of sperm and alter the phosphorylation levels of PKA substrates and PKC substrates, while the competitive inhibitors Rp-cAMP and bisindolylmaleimide (BIM) do not. Altogether, our results show that both PKA and PKC participate in sperm motility regulation through a crosstalk in the signalling pathway. These results contribute to a better understanding of the mechanisms that govern motility in sea urchin sperm.

Methcathinone decreases dopamine transporter function: Role of protein kinase C.

Methcathinone (MCAT) is a psychostimulant of abuse that can cause both persistent striatal dopaminergic and serotonergic, as well as hippocampal serotonergic, deficits. Evidence suggests that the rapid effects of stimulants that are structurally and mechanistically similar to MCAT on monoamine transporter function may contribute to the abuse liability and/or persistent monoaminergic deficits caused by these agents. Thus, effects of MCAT on 1) striatal dopamine (DA) transporter (DAT); and 2) striatal and hippocampal serotonin transporter (SERT) function, as determined in tissues from adult male rats, were assessed. As reported previously, a single administration of MCAT rapidly (within 1hr) decreases striatal [3 H]DA uptake. Similarly, incubation of rat synaptosomes with MCAT at 37℃ (but not 4˚C) decreased striatal [3 H]DA uptake. Incubation with MCAT likewise decreased [3 H]5HT but not vesicular [3 H]DA uptake. MCAT incubation in vitro was without effect on [3 H]DA uptake in striatal synaptosomes prepared from MCAT-treated rats. The decrease in [3 H]DA uptake caused by MCAT incubation: (a) reflected a decrease in Vmax , with minimal change in Km , and (b) was attenuated by co-incubation with the cell-permeable calcium chelator, N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]-1,1'-bis[(acetyloxy)methyl] ester-glycine (BAPTA-AM), as well as the non-selective protein kinase-C (PKC) inhibitors bisindolylmaleimide-1 (BIM-1) and 2-[1-3(Aminopropyl)indol-3-yl]-3(1-methyl-1H-indol-3-yl)maleimide (or Bisindolylmaleimide VIII; Ro-31-7549). Taken together, these results suggest that in vitro MCAT incubation may model important aspects of MCAT administration in vivo, and that calcium and PKC contribute to the in vitro effects of MCAT on DAT.

OSU-2S, a Novel PKC Activator, Mediates PKC Dependent Cell Death, Differentiation and Cell Cycle Arrest in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a near incurable disease with 5-year survival rates of 10% in older patients. Poor tolerance to chemotherapy, chemo resistance and high rate of relapse warrants less toxic and more effective regimens in AML. AML leads to a block in normal myeloid differentiation, leading to an accumulation of undifferentiated blasts. Thus, therapies directed towards promoting these cells to differentiate re-establishes the ability of AML cells to undergo cell death, reducing the leukemic population. Such differentiation therapy with all-trans retinoic acid (ATRA) has been successful in acute promyelocytic leukemia (APL), but not in other AML subtypes resistant to ATRA, thus presenting a promising and unexploited avenue for targeted therapies. There is accumulating evidence that Protein Kinase C (PKC) activation induces myeloid differentiation. However, since most PKC activators are either tumor promoters or have inconclusive activity, their potential for use in AML has yet to be realized. OSU-2S is a non-immunosuppressive derivative of FTY720 (a sphingosine analogue with anti-tumorigenic properties) and has previously been reported to directly activate PKC to mediate cytotoxicity, showing promising in vitro and in vivo activity against a number of leukemias and lymphomas, and other malignancies such as hepatocellular carcinoma. This impelled us to evaluate the activity of OSU-2S in causing cell death and differentiation in AML. We found that OSU-2S resulted in increased activation of PKC in HL-60 cells. In addition to effecting significant dose-dependent cytotoxicity against AML cell lines HL-60, MV4-11 and MOLM-13 [mean decrease in viability with OSU-2S in HL-60: 85±7% (p=0.08), MV4-11: 74±20% (p=0.02), MOLM-13: 60±25% (p=0.04)], and in primary samples from leukemic patients, including high risk FLT3-ITD mutated AMLs (65±8% decrease in viability with OSU-2S, p<0.0001, n=13, dose trend p<0.0001), OSU-2S caused a significant upregulation of CD11b, a cell-surface marker associated with myeloid maturation, in HL-60 and primary AML samples [(25±5% and 14±7% increase in CD11b+ cells with OSU-2S vs vehicle control in HL-60 (p =0.0126) and primary AML (p=0.043) cells respectively]. In addition, OSU-2S treated HL-60 and primary AML cells exhibited morphological features indicative of differentiation, as evidenced by lower nuclear to cytoplasmic ratio, and highly vacuolated and granulated cytoplasm. The observed differentiation activity prompted us to test if OSU-2S affected the leukemia-initiating cell population. Colony forming assays showed OSU-2S significantly reduced the number of colony forming units-leukemic (CFU-L) in primary AML blasts (52±13% decrease in CFU-L in response to OSU-2S vs vehicle control; p=0.018, n=4). OSU-2S mediated cytotoxicity was significantly abrogated in the presence of PKC inhibitor Bisindolylmaleimide (BIS) in HL-60 cell line [39±8% decrease in OSU-2S induced cytotoxicity in the presence of BIS vs control (p=0.008), n=3] as well as in primary AML cells [41±16% decrease in OSU-2S induced cytotoxicity (p=0.02), n=6], indicating that OSU-2S mediates its cytotoxic effects in AML via PKC activation.To further gain insight into the mechanism of cell death and differentiation effected by OSU-2S, we performed RNA-Sequencing analysis of total RNA isolated from MV4-11 cells treated with OSU-2S. Ingenuity Pathway Analysis on these transcripts showed significant changes in gene expression affecting cell cycle regulation and proliferation (p=10-13). Cell cycle analysis of MV4-11, HL-60 and U-937 cells by PI staining followed by flow cytometry confirmed a decrease in cells in G2/M phase of the cell cycle associated with increased cells in S but not Go/G1 phase (69.63% decrease in G2/M phase cells with OSU-2S vs vehicle control, p=0.04). Current ongoing studies in cell line-derived xenografted mouse models of AML indicate favorable therapeutic effect of OSU-2S in providing survival advantage in AML. Tumor burden monitoring by bioluminescent imaging shows reduction in tumor burden following OSU-2S treatment. Our results show promising pre-clinical efficacy of OSU-2S against AML, and warrants further studies to establish clinical efficacy as a potential therapeutic agent. Ongoing studies are also directed towards studying the involvement and role of PKC and other target molecules in the cell death and differentiation effected by OSU-2S. DisclosuresWalker:Gilead: Research Funding. Mims:Novartis: Honoraria. Chen:The Ohio State University: Patents & Royalties: OSU-2S. Byrd:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; Janssen: Research Funding; Genentech: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S. Vasu:Boehringer-Ingelheim: Research Funding; Stemline Therapeutics: Research Funding.

5-Hydroxytryptamine Modulates Maturation and Mitochondria Function of Human Oligodendrocyte Progenitor M03-13 Cells.

Inside the adult CNS, oligodendrocyte progenitor cells (OPCS) are able to proliferate, migrate and differentiate into mature oligodendrocytes (OLs) which are responsible for the production of myelin sheet and energy supply for neurons. Moreover, in demyelinating diseases, OPCs are recruited to the lesion areas where they undergo differentiation and myelin synthesis. Serotonin (5-hydroxytryptamine, 5-HT) is involved in OLs’ development and myelination, but so far the molecular mechanisms involved or the effects of 5-HT on mitochondria function have not yet been well documented. Our data show that 5-HT inhibits migration and proliferation committing cells toward differentiation in an immortalized human oligodendrocyte precursor cell line, M03-13. Migration blockage is mediated by reactive oxygen species (ROS) generation since antioxidants, such as Vit C and Cu-Zn superoxide dismutase, prevent the inhibitory effects of 5-HT on cell migration. 5-HT inhibits OPC migration and proliferation and increases OL phenotypic markers myelin basic protein (MBP) and Olig-2 via protein kinase C (PKC) activation since the inhibitor of PKC, bis-indolyl-maleimide (BIM), counteracts 5-HT effects. NOX inhibitors as well, reverse the effects of 5-HT, indicating that 5-HT influences the maturation process of OPCs by NOX-dependent ROS production. Finally, 5-HT increases mitochondria function and antioxidant activity. The identification of the molecular mechanisms underlying the effects of 5-HT on maturation and energy metabolism of OPCs could pave the way for the development of new treatments for autoimmune demyelinating diseases such as Multiple Sclerosis where oligodendrocytes are the primary target of immune attack.

Mitochondrial dysfunction increases bronchopulmonary C‐fiber excitability via PKC alpha signaling

Airway sensory nerve excitability is a key determinant of respiratory disease‐associated reflexes and sensations such as cough and dyspnea. One notable feature of peripheral terminals of sensory nerves is that they are packed with mitochondria. Mitochondrial dysfunction and subsequent oxidative stress has been reported for a variety of cell types in inflammatory diseases. We have previously shown that reactive oxygen species (ROS) produced by Antimycin A (a mitochondrial complex III Qi site inhibitor) increase excitability in bronchopulmonary nociceptive C‐fibers. This increased excitability decreases the mechanical threshold for activation and increases action potential firing in response to a P2X2/3 agonist. This Antimycin A–induced hyperexcitability is dependent on mitochondrial ROS and is blocked by intracellular antioxidants.Given that ROS are known to activate protein kinase C (PKC), we hypothesized that this increased excitability was due to PKC activation. We used dissociated vagal ganglion neurons to study activation of the PKC isoforms: alpha, beta1, beta2, delta and epsilon in response to Antimycin‐A. Cells were stained with antibodies against the specific isoforms to determine their cellular location. Of these isoforms, only PKC alpha translocated to the region of the cell membrane in response to Antimycin A treatment.We also examined the effects of Antimycin A induced ROS production on sensory nerve terminals. We recorded action potential firing from the terminals of individual bronchopulmonary C‐fibers using a mouse ex vivo lung‐vagal ganglia preparation. C‐fibers were characterized as nociceptors or non‐nociceptors based upon conduction velocity and response to transient receptor potential (TRP) channel agonists. Antimycin A–induced hyperexcitability was inhibited by the PKC inhibitor bisindolylmaleimide (BIM) I, but not by its inactive analog BIM V. Further, this BIM I inhibition occurred at a concentration that specifically blocks activity of PKC alpha over other isoforms. In conclusion, ROS evoked by mitochondrial dysfunction caused nociceptor hyperexcitability via the translocation and activation of PKC alpha.Support or Funding InformationThis work was supported by the National Heart Lung and Blood Institute in Bethesda, USA (R01‐HL119802)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Defining rules for cancer cell proliferation in TRAIL stimulation

Owing to their self-organizing evolutionary plasticity, cancers remain evasive to modern treatment strategies. Previously, for sensitizing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant human fibrosarcoma (HT1080), we developed and validated a dynamic computational model that showed the inhibition of protein kinase (PK)C, using bisindolylmaleimide (BIS) I, enhances apoptosis with 95% cell death. Although promising, the long-term effect of remaining ~ 5% cells is a mystery. Will they remain unchanged or are they able to proliferate? To address this question, here we adopted a discrete spatiotemporal cellular automata model utilizing simple rules modified from the famous “Conway’s game of life”. Based on three experimental initializations: cell numbers obtained from untreated (high), treatment with TRAIL only (moderate), and treatment with TRAIL and BIS I (low), the simulations show cell proliferation in time and space. Notably, when all cells are fixed in their initial space, the proliferation is rapid for high and moderate cell numbers, however, slow and steady for low number of cells. However, when mesenchymal-like random movement was introduced, the proliferation becomes significant even for low cell numbers. Experimental verification showed high proportion of mesenchymal cells in TRAIL and BIS I treatment compared with untreated or TRAIL only treatment. In agreement with the model with cell movement, we observed rapid proliferation of the remnant cells in TRAIL and BIS I treatment over time. Hence, our work highlights the importance of mesenchymal-like cellular movement for cancer proliferation. Nevertheless, re-treatment of TRAIL and BIS I on proliferating cancers is still largely effective.

Quercetin Increases MUC2 and MUC5AC Gene Expression and Secretion in Intestinal Goblet Cell-Like LS174T via PLC/PKCα/ERK1-2 Pathway.

The main dietary flavonoid quercetin, is known to preserve the integrity of gastrointestinal barrier and to have anti-inflammatory, anti-cancer, anti-fibrotic, and other beneficial properties. Many of the biological effects of quercetin appear to be associated to the modulation of cell signaling pathways, rather than to its antioxidant activity. In spite of the large number of data available on the molecular and cellular mechanisms by which quercetin exerts its biological effects, including protection of intestinal barrier function, there is a lack of data about the role of this substance on the expression and/or the secretion of mucins released by intestinal goblet cells. Here we investigated the effects of quercetin on the secretion and the gene expression of the main intestinal gel-forming mucins, MUC2 and MUC5AC, and the signaling mechanisms underlined, in human intestinal goblet cell-like LS174T. We found that quercetin increases intracellular Ca2+ levels and induces MUC2 and MUC5AC secretion in a Ca2+-dependent manner. Quercetin also induces mRNA levels of both secretory mucins. Quercetin stimulation of LS174T cells increases phosphorylation levels of extracellular signal regulated kinase (ERK)1-2 and protein kinase C (PKC) α and the induction of MUC2 and MUC5AC secretion and mRNA relies on phospholipase C (PLC), PKC, and ERK1-2 signaling pathways since the PLC inhibitor U73122, the PKC inhibitor bisindolylmaleimide (BIM) and the ERK1-2 pathway inhibitor PD98059, all revert the stimulatory effects of quercetin. We also demonstrated that the induction of mucin gene expression by quercetin is not limited to goblet cells. Indeed, quercetin induces mRNA levels of MUC2 and MUC5AC via PKCα/ERK1-2 pathway also in the human intestinal epithelial Caco-2 cells. These data highlight a novel mechanism thereby quercetin, regulating the secretory function of intestinal goblet cells and mucin levels in enterocytes may exert its protective effects on intestinal mucosal barrier.

Mitochondrial ROS activates PKC alpha translocation to the membrane of vagal sensory neurons.

Hyperexcitability of airway sensory nerves can trigger disease‐associated reflexes resulting in cough and dyspnea. We have observed that mitochondrial ROS production increases hyperexcitability in nociceptive bronchopulmonary C‐fibers. We also found that this increased excitability can be blocked by the PKC inhibitor bisindolylmaleimide (BIM) I, but not the inactive PKC analog BIM V. In previous work we have shown, using a pan‐PKC antibody, that mitochondrial ROS production via Antimycin A (AntA; mitochondrial complex III Qi site inhibitor) causes translocation to the cell membrane. However, there are several PKC isoforms expressed in nociceptive neurons. We wanted to determine which isoforms were present, their distribution in nociceptive cells and whether specific isoforms were activated by AntA. Studies looking at mRNA expression have highlighted PKC alpha, beta1, beta2, delta and epsilon as abundant in nociceptors so our studies have focused on these particular isoforms. Sensory neuron cell bodies were isolated from mouse vagal ganglia. To identify nociceptive cells, we used a mouse strain that co‐expresses a red fluorescent protein (tdTomato) with the Transient Receptor Potential V1 ion channel. Cells were treated for 2 min with: vehicle, 10 μM AntA or 300 nM PMA then immediately fixed. We performed immunocytochemistry to determine cellular location of different isoforms. Of the TRPV1 expressing nociceptive cells, more than 50% were positive for any given isoform of PKC. All isoforms translocated to the membrane in response to PMA treatment. However, only PKC alpha exhibited translocation to the membrane in response to AntA treatment. In the case of PKC delta, AntA appeared to cause translocation of this isoform to the nucleus. Our experiments suggest that nociceptor hyperexcitability, in response to ROS produced via mitochondrial dysfunction, may occur through the translocation and activation of the PKC alpha isoform.Support or Funding InformationThis work was supported by the National Heart Lung and Blood Institute in Bethesda, USA(R01‐HL119802)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neurotoxic Dopamine Transporter Substrates Differentially Affect DAT Regulation, Uptake, and Reverse Transport

Synaptic action of the neurotransmitter dopamine (DA) is primarily terminated by reuptake into presynaptic dopaminergic neurons via dopamine transporters (DATs). DATs are integral plasmalemmal proteins whose reuptake function is tightly coordinated by interplay between the post translational modifications phosphorylation and palmitoylation. The psychostimulant amphetamine (AMPH) is a DAT substrate known to empty sequestered DA from vesicles and induce protein kinase C (PKC)‐dependent DAT phosphorylation, resulting in reverse transport of DA (efflux) greatly increased DA signaling. Elevated DA concentrations similar to those produced by AMPH create neurotoxic conditions as DA oxidation produces free radicals and hinders mitochondrial respiration, thus strict spatial and temporal control of cytosolic and synaptic DA is essential for neuronal health. DA induced oxidative stress is an important pathogenic mediator in Parkinson's disease (PD), and animal models of PD are generated by the neurotoxins 1‐methyl‐4‐phenylpyridinium ion (MPP+) and 6‐hydroxydopamine (6OHDA) which selectively target DAT‐expressing neurons, are translocated to the cytosol by DAT, and perturb the oxidative balance within the cell, producing nigrostriatal DA neuron death observed in PD. It is therefore intriguing to investigate DAT‐involved mechanisms in terms of PD etiology as atypical DAT function could contribute to DA oxidative stress and is hypothesized to play a role in other neurological disorders including attention deficit/hyperactivity disorder (ADHD) and addiction. DAT expression occurs around synaptic terminals, along axons, and on dendritic spines, and efflux via DATs located on dendrites in the substantia nigra has been shown to diminish dopaminergic signaling. It is possible, therefore, for aberrant regulation and localization of DA clearance and DAT‐mediated DA efflux to profoundly influence the excitability of neurons and play a significant role in neurodegeneration in the progression of PD. This is even more interesting in the light of recent studies revealing DAT polymorphisms in patients diagnosed with a psychiatric disorder or early onset PD that display an elevated basal DA efflux phenotype. In this study, we are investigating the effect of DAT substrates and PD‐inducing neurotoxins on DAT phosphorylation, palmitoylation, down‐regulation, and reverse transport to better understand aberrant DAT mechanisms which may contribute the onset of PD. Our findings thus far indicate MPP+ and 6OHDA differentially alter DAT function. 6OHDA inhibits basal and attenuates PMA‐induced DAT phosphorylation, does not significantly reduce DAT function, and decreases DA efflux. MPP+ does not change basal or PMA stimulated DAT phosphorylation yet it induces significant DA efflux which may be an additional means by which MPP+ produces neurodegeneration. This MPP+ induced efflux was not blunted by pretreatment with the PKC inhibitor bisindolylmaleimide (BIM). Although AMPH‐induced efflux was blunted after BIM treatment, significant efflux remained suggesting mechanisms independent of PKC mediated DAT phosphorylation for the stimulation of DA efflux.Support or Funding InformationSupported by the National Institute on Drug Abuse Grant DA 031991 JDF, P20 RR017699 (to U.N.D.) from the COBRE program of the National Center for Research Resources, and P20 RR016741 (to U.N.D.) from the INBRE program of the National Center for Research Resources.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The differential roles of PEA15 phosphorylations in reactive astrogliosis and astroglial apoptosis following status epilepticus

Up to this day, the roles of PEA15 expression and its phosphorylation in seizure-related events have not been still unclear. In the present study, we found that PEA15 was distinctly phosphorylated in reactive astrocytes and apoptotic astrocytes in the rat hippocampus following LiCl-pilocarpine-induced status epilepticus (SE, a prolonged seizure activity). PEA15-serine (S) 104 phosphorylation was up-regulated in reactive astrocytes following SE, although PEA15 expression and its S116 phosphorylation were unaltered. Bisindolylmaleimide (BIM), a protein kinase C (PKC) inhibitor, attenuated SE-induced reactive astrogliosis, but phorbol 12-myristate 13-acetate (PMA, a PKC activator) aggravated it. Unlike reactive astrocytes, PEA15-S116 phosphorylation was reduced in apoptotic astrocytes. However, PEA15 expression and its S104 phosphorylation were unchanged in apoptotic astrocyte. Neither BIM nor PMA affected SE-induced astroglial apoptosis. PEA15 expression and its phosphorylations were not relevant to SE-induced CA1 neuronal death. These findings indicate that PEA15-S104 and S116 phosphorylations may play a role in reactive astrogliosis and prevention of astroglial apoptosis, respectively. Therefore, we suggest that the selective manipulation of PEA15 phosphorylations may regulate apoptotic and/or proliferative signals in astrocytes.

Functional expression and pharmaceutical efficacy of cardiac-specific ion channels in human embryonic stem cell-derived cardiomyocytes

Cardiomyocytes differentiated from human pluripotent stem cells provide promising tools for screening of cardiotoxic drugs. For evaluation of human pluripotent stem cell-derived cardiomyocytes for cardiotoxicity test, in the present study, human embryonic stem cells (hESCs) were differentiated to cardiomyocytes, followed by metabolic selection to enrich the differentiated cardiomyocytes. The highly purified hESC-derived cardiomyocytes (hESC-CMs) expressed several cardiomyocyte-specific markers including cTnT, MLC2a, and α-SA, but not pluripotency markers, such as OCT4 and NANOG. Patch clamp technique and RT-PCR revealed the expression of cardiomyocyte-specific Na+, Ca2+, and K+ channels and cardiac action potential in hESC-CMs. To explore the potential use of hESC-CMs as functional cardiomyocytes for drug discovery and cardiotoxicity screening, we examined the effects of bisindolylmaleimide (BIM) (I), which inhibits native cardiac Ca2+ channels, on the Ca2+ channel activity of hESC-CMs. We observed a similar response for the BIM (I)-induced modulation of Ca2+ channels between hESC-CMs and native cardiomyocytes through L-type Ca2+ channel current. These results suggest that hESC-CMs can be useful for evaluation of pharmaceutical efficacy and safety of novel drug candidate in cardiac research.

Synthesis and properties of novel heat-resistant fluorescent conjugated polymers with bisindolylmaleimide

Conjugated polymers with bisindolylmaleimide (BIM) backbone are obtained by the condensation polymerization of methyl and octanyl N-substituted BIMs with 4,4'-difluoro-diphenylsulfone and 4,4'-difluoro-diphenylketone. The structures of polymers are confirmed by FTIR and NMR spectroscopy. The polymers exhibit both high glass transition temperatures (Tg>175°C) and high decomposition temperatures (T5>395°C). Meanwhile, The UV–vis absorption and fluorescence spectra of the polymers are similar to the corresponding substituted BIMs. The quantum chemistry calculations indicate that the first excited states of polymers are mostly contributed by BIM structures.

Ligands of peroxisome proliferator-activated receptor-alpha promote glutamate transporter-1 endocytosis in astrocytes

Astrocytes, a stellate-shape glial population in the central nervous system (CNS), maintain glutamate homeostasis in adult CNS by undergoing glutamate uptake at the synapse through their glutamate transporter-1 (GLT-1). Peroxisome proliferator-activated receptor-α (PPARα) can be activated by endogenous saturated fatty acids to regulate astrocytic lipid metabolism and functions. However, it is unclear if PPARα can exert the regulatory action on GLT-1 expression in astrocytes. This study showed that treatment with palmitic acid (PA) and the other two PPARα agonists (GW 7647 and WY 14,643) caused no change in the morphology of astrocytes, whereas membranous GLT-1 protein levels in astrocytes were significantly decreased by PA and PPARα agonists. Through lentivirus-mediated overexpression of GLT-1 tagged with red fluorescent protein (GLT-1-RFP), we also observed that GLT-1-RFP puncta in the processes of astrocytes were inhibited by the PPARα agonists. This reduction was prevented by the addition of the PPARα antagonist, GW6471. GLT-1-RFP was co-localized to the early endosome marker–EEA1 in astrocytes treated with the PPARα agonists. Moreover, PPARα-induced inhibition in membranous GLT-1 expression was abolished by the addition of dynamin inhibitor (dynasore). Furthermore, the co-treatment of astrocytes with PPARα agonists and dynasore, or with PPARα agonists and protein kinase C (PKC) inhibitor bis-indolylmaleimide 1 (BIS1), prevented the endocytosis of GLT-1-RFP. Based on the results, we conclude that the PPARα agonists increased GLT-1 endocytosis in astrocytes possibly through the PKC signaling pathway. In addition, our findings provide important information of PPARα involvement in the downregulation of astrocytic glutamate uptake via the promoted GLT-1 endocytosis.

Reactive Oxygen Species Derived from NOX3 and NOX5 Drive Differentiation of Human Oligodendrocytes.

Reactive oxygen species (ROS) are signaling molecules that mediate stress response, apoptosis, DNA damage, gene expression and differentiation. We report here that differentiation of oligodendrocytes (OLs), the myelin forming cells in the CNS, is driven by ROS. To dissect the OL differentiation pathway, we used the cell line MO3-13, which display the molecular and cellular features of OL precursors. These cells exposed 1–4 days to low levels of H2O2 or to the protein kinase C (PKC) activator, phorbol-12-Myristate-13-Acetate (PMA) increased the expression of specific OL differentiation markers: the specific nuclear factor Olig-2, and Myelin Basic Protein (MBP), which was processed and accumulated selectively in membranes. The induction of differentiation genes was associated with the activation of ERK1-2 and phosphorylation of the nuclear cAMP responsive element binding protein 1 (CREB). PKC mediates ROS-induced differentiation because PKC depletion or bis-indolyl-maleimide (BIM), a PKC inhibitor, reversed the induction of differentiation markers by H2O2. H2O2 and PMA increased the expression of membrane-bound NADPH oxidases, NOX3 and NOX5. Selective depletion of these proteins inhibited differentiation induced by PMA. Furthermore, NOX5 silencing down regulated NOX3 mRNA levels, suggesting that ROS produced by NOX5 up-regulate NOX3 expression. These data unravel an elaborate network of ROS-generating enzymes (NOX5 to NOX3) activated by PKC and necessary for differentiation of OLs. Furthermore, NOX3 and NOX5, as inducers of OL differentiation, represent novel targets for therapies of demyelinating diseases, including multiple sclerosis, associated with impairment of OL differentiation.

A crystalline bisindolylmaleimide with strong solid-state fluorescence of red color and its analogous cross-linked polymer without fluorescence

A crystalline bisindolylmaleimide (BIM) 3 with strong fluorescence in the solid state was prepared and studied. Its analogous cross-linked polymer 4 with the same BIMs fluorophore was designed and characterized. The investigation of the covalent bonding networking effect on the solid-state fluorescence was carried out by the comparison between 3 and 4.