Botulinum neurotoxin dose-dependently inhibits release of neurosecretory vesicle-targeted luciferase from neuronal cells

Abstract

SummaryBotulinum 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.

Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins

“…The generation of the stably transfected human neuroblastoma cell line SIMA hPOMC1-26-GLuc has been described previously [ 16 ]. Cells were cultured in RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 10% ( v / v ) heat-inactivated fetal calf serum (FCS, Biochrom, Berlin, Germany), while 2 mM stable l -alanyl- l -glutamine and penicillin (100 U/mL)/streptomycine (100 µg/mL) (Biochrom, Berlin, Germany) were used as antibiotics.…”

“…Recently, we developed a cell-based assay that determines the BoNT activity by quantifying the inhibition of the release of a luciferase, which has been redirected into neurosecretory vesicles. This can act as a surrogate for the inhibition of neurotransmitter release [ 16 ]. In theory, this assay should overcome the limitations of the other currently established in vitro or cell-based assays as it should be suitable for all BoNT serotypes and for the tetanus toxin, which also cleaves the VAMP protein.…”

Cell-Based Reporter Release Assay to Determine the Activity of Calcium-Dependent Neurotoxins and Neuroactive Pharmaceuticals

“…Muscarinic acetylcholine receptor: In two former projects, release of the reporter enzyme GLuc was stimulated by a high K + -depolarization buffer and entry of extracellular Ca 2+ into the reporter cell line [10,11]. One aim of this study was to analyze if GLuc can also be released by a Ca 2+ -increase from intracellular pools.…”

“…From these vesicles, GLuc was released upon depolarization of the cells into the cell culture supernatant together with neurotransmitters. In proof of principle, it was recently shown that the depolarizationdependent release was efficiently inhibited by BoNT/A and to a lesser extent by BoNT/B and tetanus toxin [10,11]. The goal of this study was to analyze whether this assay is also suitable to detect the activity of other neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release.…”

Technologies for Detecting Botulinum Neurotoxins in Biological and Environmental Matrices

“…Hong et al [53] have also developed a similar assay using a motor neuron-like continuous cell line. Pathe-Neuschäfer-Rube et al [54] developed a N-terminal tagged luciferase-expressing neuronal cell line. Luciferase is released from these transfected cells during depolarization, which is blocked by botulinum toxin.…”