Investigating the Role of Two-Pore Channel 2 (TPC2) in Zebrafish Neuromuscular Development

2020 ◽  
pp. 103-134
Author(s):  
Sarah E. Webb ◽  
Jeffrey J. Kelu ◽  
Andrew L. Miller
Keyword(s):  
Pore Channel ◽  
Neuromuscular Development

scholarly journals Role of the Putative NAADP Receptor, Two-Pore Channel 2, in Ventricular Myocyte Responses to Isoproterenol

2010 ◽  
Vol 98 (3) ◽  
pp. 100a
Author(s):  
Laura C. Elson ◽  
William D. Owen ◽  
Arie R. Gafson ◽  
John Parrington ◽  
Antony Galione ◽  
...  
Keyword(s):  
Pore Channel ◽  
Ventricular Myocyte

scholarly journals Blocking Lysosomal Two-Pore Channel 2 Function Inhibits Proliferation of Multidrug Resistant Leukemia Cells and Sensitizes Them to Vincristine Treatment

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2081-2081
Author(s):  
Martin Müller ◽  
Franz Geisslinger ◽  
Susanne Gerndt ◽  
Ramona Schütz ◽  
Yu-Kai Chao ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Patch Clamp ◽  
Mrna Expression ◽  
Treatment Response ◽  
Cell Line ◽  
Doubling Time ◽  
Multidrug Resistant ◽  
Pore Channel ◽  
Increased Sensitivity

Introduction Two-pore channel 2 (TPC2) represents an exceptional ion channel due to its localization on lysosomes1. TPC2 is overexpressed in several cancer types, e.g. in prostate cancer, and its expression is associated with poor survival probability2. To date, a role of TPC2 in leukemia has not been investigated. Pharmacologic inhibition of TPC2 can be performed with tetrandrine (Tet), a bisbenzylisoquinoline alkaloid3. However, its use is restricted due to unfavorable toxicity in animal studies4. In our study, we aim to elucidate the role of TPC2 function in multidrug resistant leukemia and, concurrently, identify efficacious TPC2 inhibitors by screening a library of synthetic bisbenzylisoquinoline derivatives (BBIQDs). Results Firstly, via qPCR analysis, we detected a 2-fold increased TPC2 mRNA expression in a vincristine-resistant (VCR-R) CEM (VCR-R CEM, T-ALL) cell line, compared with the parental CCRF-CEM cell line. Secondly, we knocked out TPC2 in VCR-R CEM cells using the CRISPR-Cas9 system to investigate its involvement in cell growth and treatment response. By studying cell proliferation over a period of six days, we found that the doubling time of TPC2-deficient cells was increased to 30 h, compared with 24 h for the wildtype (wt) cell line. Additionally, treatment of TPC2 knockout (ko) cells with VCR resulted in increased sensitivity versus wt, as indicated by proliferation (IC50 wt: 3.3 µM, IC50 ko: 1.6 µM, 72 h) and apoptosis (EC50 wt: 3.0 µM, EC50 ko: 1.7 µM, 48 h) assays. Next, a library of BBIQDs was synthesized and screened for the ability to inhibit TPC2 function using the whole endolysosomal patch clamp method. Our small molecule hits SG-005 and SG-094 inhibited TPC2 current density by 50% and 70%, respectively, giving novel TPC2 inhibitors with either similar or even increased potency, compared to Tet (50%). Treatment of VCR-R CEM cells with Tet, SG-005 and 94 effectively inhibited proliferation (IC50: 5-15 µM, 48 h). Toxicity was assessed by propidium iodide exclusion assays using Peripheral Blood Mononuclear cells (PBMCs, n=3 healthy donors), indicating reduced toxicity of SG-094 (<5% dead cells vs. 25% dead cells for Tet and SG-005, 20 µM, 48 h treatment). Additionally, SG-094 was well tolerated in vivo when applied to a mouse model (90 nmol/kg/d on three consecutive days). Remarkably, combination of VCR (0.01 and 0.1 µM) with Tet, SG-005 or 94 (1 and 5 µM) synergistically increased treatment response of VCR-R CEM cells (wt) and of B ALL patient-derived xenograft (PDX) cells from a relapse patient (Bliss values: 1.1-2.4). Methods CellTiter-Blue® cell proliferation assays, qPCR mRNA expression, propidium iodide exclusion and analysis of apoptosis were performed as described by the manufacturers. Whole endolysosomal patch clamp, analysis of specific apoptosis of PDX cells and generation of the TPC2 ko model using the CRISPR-Cas9 system were performed as described previously3, 5, 6. Analysis of doubling time was performed by counting cells daily using trypan blue staining and a Vi-CELL XR device as indicated by the manufacturer. Conclusion Taken together, we detected an upregulated mRNA expression of endolysosomal TPC2 in VCR-R CEM cells, indicating that it represents a potential target for therapeutic intervention for difficult-to-treat leukemia phenotypes. Furthermore, we could show that genetic ablation of the ion channel results in a reduced proliferation rate and an increased sensitivity towards VCR treatment. Finally, we successfully identified pharmacologic TPC2 inhibitors which show antiproliferative effects when applied as monotherapy and synergistically enhance treatment response of VCR-R CEM cells and PDX cells when combined with VCR. References S. Patel, B. S. Kilpatrick, Biochim Biophys Acta Mol Cell Res1865, 1678-1686 (2018). F. Li, J. P. Ji, Y. Xu, R. L. Liu, Clin Transl Oncol, (2019). O. N. Nguyen et al., Cancer Res77, 1427-1438 (2017). H. Jin et al., Chem Res Toxicol24, 2142-2152 (2011). F. Koczian et al., Haematologica104, 546-555 (2019). F. A. Ran et al., Nat Protoc8, 2281-2308 (2013). Disclosures No relevant conflicts of interest to declare.

scholarly journals Estimate of ULF electromagnetic noise caused by a fluid flow during seismic or volcano activity

2014 ◽  
Vol 2 (10) ◽  
pp. 6475-6497
Author(s):  
V. V. Surkov ◽  
V. A. Pilipenko
Keyword(s):  
Electromagnetic Emission ◽  
Theoretical Models ◽  
Pore Channel ◽  
Electromagnetic Noise ◽  
Rock Permeability ◽  
Magma Flow ◽  
Observational Technique ◽  
Water Saturated ◽  
Favorable Conditions

Abstract. The elaboration of theoretical models, even oversimplified, capable to estimate an expected electromagnetic effect during earthquake preparation process is not less important than the advancement of observational technique to detect seismic-related electromagnetic emission. Here possible mechanisms of ULF electromagnetic noise associated with seismic or volcanic activity are discussed. The electrokinetic (EK) and magnetohydrodynamic (MHD) effects due to an irregular flow of conducting rock fluid or magma flow are being revised. The conventional theory of EK effect in a water-saturated rocks has been advanced by consideration of elliptic-shaped channels. A contribution of both mechanisms to observed ULF signal on the ground is shown to be dependent on the pore channel size/rock permeability. Estimates of magnetic and telluric perturbations caused by magma motion along a volcano throat indicate on the important role of the surrounding rock conductivity. These estimates have proven that the mechanisms under consideration are able to generate ULF electromagnetic emission which could be detected by modern magnetometers under favorable conditions.

scholarly journals ErbB4 tyrosine kinase inhibition impairs neuromuscular development in zebrafish embryos

2019 ◽  
Vol 30 (2) ◽  
pp. 209-218 ◽  
Author(s):  
Ilkka Paatero ◽  
Ville Veikkolainen ◽  
Matias Mäenpää ◽  
Etienne Schmelzer ◽  
Heinz-Georg Belting ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Wide Spectrum ◽  
Erbb Receptors ◽  
Limited Information ◽  
Targeted Mutation ◽  
Neuromuscular Development

Tyrosine kinase inhibitors are widely used in the clinic, but limited information is available about their toxicity in developing organisms. Here, we tested the effect of tyrosine kinase inhibitors targeting the ErbB receptors for their effects on developing zebrafish ( Danio rerio) embryos. Embryos treated with wide-spectrum pan-ErbB inhibitors or erbb4a-targeting antisense oligonucleotides demonstrated reduced locomotion, reduced diameter of skeletal muscle fibers, and reduced expression of muscle-specific genes, as well as reduced motoneuron length. The phenotypes in the skeletal muscle, as well as the defect in motility, were rescued both by microinjection of human ERBB4 mRNA and by transposon-mediated muscle-specific ERBB4 overexpression. The role of ErbB4 in regulating motility was further controlled by targeted mutation of the endogenous erbb4a locus in the zebrafish genome by CRISPR/Cas9. These observations demonstrate a potential for the ErbB tyrosine kinase inhibitors to induce neuromuscular toxicity in a developing organism via a mechanism involving inhibition of ErbB4 function.

scholarly journals TPC1 deficiency or blockade augments systemic anaphylaxis and mast cell activity

2020 ◽  
Vol 117 (30) ◽  
pp. 18068-18078 ◽  
Author(s):  
Elisabeth Arlt ◽  
Marco Fraticelli ◽  
Volodymyr Tsvilovskyy ◽  
Wiebke Nadolni ◽  
Andreas Breit ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Ex Vivo ◽  
Secretory Granules ◽  
Cell Activity ◽  
New Drugs ◽  
Pore Channel ◽  
Systemic Anaphylaxis

Mast cells and basophils are main drivers of allergic reactions and anaphylaxis, for which prevalence is rapidly increasing. Activation of these cells leads to a tightly controlled release of inflammatory mediators stored in secretory granules. The release of these granules is dependent on intracellular calcium (Ca2+) signals. Ca2+release from endolysosomal compartments is mediated via intracellular cation channels, such as two-pore channel (TPC) proteins. Here, we uncover a mechanism for how TPC1 regulates Ca2+homeostasis and exocytosis in mast cells in vivo and ex vivo. Notably, in vivo TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis, reflected by increased drop in body temperature, most likely due to accelerated histamine-induced vasodilation. Ex vivo, mast cell-mediated histamine release and degranulation was augmented upon TPC1 inhibition, although mast cell numbers and size were diminished. Our results indicate an essential role of TPC1 in endolysosomal Ca2+uptake and filling of endoplasmic reticulum Ca2+stores, thereby regulating exocytosis in mast cells. Thus, pharmacological modulation of TPC1 might blaze a trail to develop new drugs against mast cell-related diseases, including allergic hypersensitivity.

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