@misc{NeuschaeferRubePatheNeuschaeferRubePueschel2022,
  author    = {Neusch{\"a}fer-Rube, Frank and Pathe-Neusch{\"a}fer-Rube, Andrea and P{\"u}schel, Gerhard Paul},
  title     = {Discrimination of the Activity of Low-Affinity Wild-Type and High-Affinity Mutant Recombinant BoNT/B by a SIMA Cell-Based Reporter Release Assay},
  series = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe},
  journal   = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe},
  publisher = {Universit{\"a}tsverlag Potsdam},
  address   = {Potsdam},
  issn      = {1866-8372},
  doi       = {10.25932/publishup-55803},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-558032},
  pages     = {1 -- 11},
  year      = {2022},
  abstract  = {Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data.},
  language  = {en}
}
@article{NeuschaeferRubePatheNeuschaeferRubePueschel2022,
  author    = {Neusch{\"a}fer-Rube, Frank and Pathe-Neusch{\"a}fer-Rube, Andrea and P{\"u}schel, Gerhard Paul},
  title     = {Discrimination of the activity of low-affinity wild-type and high-affinity mutant recombinant BoNT/B by a SIMA cell-based reporter release assay},
  series = {Toxins},
  volume    = {14},
  journal   = {Toxins},
  edition   = {1},
  publisher = {MDPI},
  address   = {Basel, Schweiz},
  issn      = {2072-6651},
  doi       = {10.3390/toxins14010065},
  pages     = {1 -- 11},
  year      = {2022},
  abstract  = {Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data.},
  language  = {en}
}
@misc{PatheNeuschaeferRubeNeuschaeferRubePueschel2021,
  author    = {Pathe-Neusch{\"a}fer-Rube, Andrea and Neusch{\"a}fer-Rube, Frank and P{\"u}schel, Gerhard Paul},
  title     = {Cell-based reporter release assay to determine the activity of calcium-dependent neurotoxins and neuroactive pharmaceuticals},
  series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe},
  journal   = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe},
  number    = {1139},
  issn      = {1866-8372},
  doi       = {10.25932/publishup-50322},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-503225},
  pages     = {15},
  year      = {2021},
  abstract  = {The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.},
  language  = {en}
}
@article{PatheNeuschaeferRubeNeuschaeferRubePueschel2021,
  author    = {Pathe-Neusch{\"a}fer-Rube, Andrea and Neusch{\"a}fer-Rube, Frank and P{\"u}schel, Gerhard Paul},
  title     = {Cell-based reporter release assay to determine the activity of calcium-dependent neurotoxins and neuroactive pharmaceuticals},
  series = {Toxins / Molecular Diversity Preservation International (MDPI)},
  volume    = {13},
  journal   = {Toxins / Molecular Diversity Preservation International (MDPI)},
  number    = {4},
  publisher = {MDPI},
  address   = {Basel},
  issn      = {2072-6651},
  doi       = {10.3390/toxins13040247},
  pages     = {13},
  year      = {2021},
  abstract  = {The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.},
  language  = {en}
}
@article{PatheNeuschaeferRubeNeuschaeferRubeGenzetal.2015,
  author    = {Pathe-Neuschaefer-Rube, Andrea and Neuschaefer-Rube, Frank and Genz, Lara and P{\"u}schel, Gerhard Paul},
  title     = {Botulinum Neurotoxin Dose-Dependently Inhibits Release of Neurosecretory Vesicle-Targeted Luciferase from Neuronal Cells},
  series = {Alternatives to animal experimentation : ALTEX ; a journal for new paths in biomedical science},
  volume    = {32},
  journal   = {Alternatives to animal experimentation : ALTEX ; a journal for new paths in biomedical science},
  number    = {4},
  publisher = {Springer},
  address   = {Heidelberg},
  issn      = {1868-596X},
  pages     = {297 -- 306},
  year      = {2015},
  abstract  = {Botulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons and thereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments and, more frequently, for aesthetic medical interventions. Botulinum toxin for pharmacological applications is isolated from bacterial cultures. Due to partial denaturation of the protein, the specific activity of these preparations shows large variations. Because of its extreme potential toxicity, pharmacological preparations must be carefully tested for their activity. For the current gold standard, the mouse lethality assay, several hundred thousand mice are killed per year. Alternative methods have been developed that suffer from one or more of the following deficits: In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic and cell based immunological assays are specific for just one of the different serotypes. The current study takes a completely different approach that overcomes these limitations: Neuronal cell lines were stably transfected with plasmids coding for luciferases of different species, which were N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The depolarization-dependent release was efficiently inhibited by botulinum toxin in a concentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay might thus be an alternative to the mouse lethality assay and the immunological assays already in use.},
  language  = {en}
}