Enhanced sensitivity to Botulinum type A neurotoxin of human neuroblastoma SH-SY5Y cells after differentiation into mature neuronal cells

Rasetti-Escargueil, Christine; Machado, Carolina Barcellos; Preneta-Blanc, R.; Fleck, Roland A.; Sesardic, Dorothea

doi:10.1504/tbj.2011.041814

Cited by 11 publications

(14 citation statements)

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“…Several cell lines (PC12, Neuro-2a, SH-SY5Y) demonstrate an increased sensitivity to BoNT/A1 when pre-loaded with trisialoganglioside GT1b for 24 h prior to toxin exposure [15,16,21]. To determine if NG108-15 cells also show an increased sensitivity to BoNT/A1 after similar pre-treatment, cells previously differentiated in culture medium containing RA and PUR were exposed to 50 μg/ml of GT1b for 24 hours prior to BoNT/A exposure for 48 h. GT1b-pretreated cells were ~1.5 times more sensitive to BoNT/A1 than untreated cells, with EC 50 values of about ~12 and ~17 LD 50 U respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, most of them lack the sensitivity necessary to compete with the commonly used mouse bioassay (MBA) [25,26], which detects low pM amounts of BoNT/A. Recent research efforts have developed protocols to optimize differentiation protocols to maximize BoNT/A sensitivity by using serum-free medium and adding neuronal growth factor (NGF) [18], retinoic acid (RA) [21], and/or addition of trisiaologanglioside GT1b [15,16,19,21,24]. The most sensitive cell-based assay using a continuous cell line published to date is using the differentiated SH-SY5Y cell line, with an EC 50 value of ~35 pM as measured by noradrenaline release [21].…”

Section: Introductionmentioning

confidence: 99%

“…Recent research efforts have developed protocols to optimize differentiation protocols to maximize BoNT/A sensitivity by using serum-free medium and adding neuronal growth factor (NGF) [18], retinoic acid (RA) [21], and/or addition of trisiaologanglioside GT1b [15,16,19,21,24]. The most sensitive cell-based assay using a continuous cell line published to date is using the differentiated SH-SY5Y cell line, with an EC 50 value of ~35 pM as measured by noradrenaline release [21]. Allergan has filed a patent for the use of SiMa cells differentiated with retinoic acid and GT1B with their product (Onabotulinumneurotoxin) with a reported sensitivity in the low pM range, comparable to the sensitivity of the MBA [24].…”

Section: Introductionmentioning

confidence: 99%

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Model for studying Clostridium botulinum neurotoxin using differentiated motor neuron-like NG108-15 cells

Whitemarsh¹,

Pier²,

Tepp³

et al. 2012

Biochemical and Biophysical Research Communications

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Cancerous cell lines have traditionally shown low sensitivity to laboratory or pharmaceutical preparations of botulinum neurotoxin. The work presented here demonstrates that the mouse neuroblastoma/ rat glioma hybrid cell line NG108-15 is capable of more sensitively detecting BoNT/A1 than any cell line previously described. This cell line has previously been described to have motor neuron like characteristics, therefore making it a good model to study BoNTs. Differentiation of NG108-15 cells in serum-free medium containing retinoic acid and purmorphamine dramatically increased sensitivity of the neurons to BoNT/A (EC50 = ~16 LD50 U). Additional pre-treatment with triasialoganglioside GT1B prior to toxin exposure reduced the EC50 further to ~11 LD50 U. Co-culture of the neurons with C2C12 myotubes also significantly increased BoNT/A sensitivity of NG108-15 cells (EC50 = 26 U) in the absence of differentiation factors.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Model for studying Clostridium botulinum neurotoxin using differentiated motor neuron-like NG108-15 cells

Whitemarsh¹,

Pier²,

Tepp³

et al. 2012

Biochemical and Biophysical Research Communications

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“…For cell-based SNAP-25 activity assay, cell lysates were tested for activity of rLC-TD/A by conducting an immunoblot for detection of cleaved SNAP-25 as described with modifications [23]. Briefly, the SH-SY5Y neuroblastoma cells (ATCC, Manassas, VA) were routinely grown and passaged in 25 cm 2 culture flasks.…”

Section: Methodsmentioning

confidence: 99%

High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics

et al. 2017

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Botulinum neurotoxins (BoNTs) are the most toxic proteins known to cause flaccid muscle paralysis as a result of inhibition of neurotransmitter release from peripheral cholinergic synapses. BoNT type A (BoNT/A) is a 150 kDa protein consisting of two major subunits: light chain and heavy chain. The light chain is required for the catalytic activity of neurotoxin, whereas the C and N terminal domains of the heavy chain are required for cell binding, and translocation of light chain across the endosome membranes, respectively. To better understand the structural and functional aspects of BoNT/A intoxication we report here the development of high yield Escherichia coli expression system (2 to 20-fold higher yield than the value reported in the literature) for the production of recombinant light chain-translocation domain (rLC-TD/A) module of BoNT/A which is catalytically active and translocation competent. The open reading frame of rLC-TD/A was PCR amplified from deactivated recombinant BoNT/A gene (a non-select agent reagent), and was cloned using pET45b (+) vector to express in Escherichia coli cells. The purification procedure included a sequential order of affinity chromatography, trypsinization, and anion exchange column chromatography. We were able to purify >95 % pure, catalytically active and structurally well-folded protein. Comparison of enzyme kinetics of purified LC-TD/A to full-length toxin and rLC/A (recombinant light chain A) suggest that the affinity for the substrate is in between endopeptidase domain and botulinum toxin. The potential application of the purified protein has been discussed in toxicity and translocation assays.

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“…Other methods that determine endogenous neurotransmitter release include direct measurement by HPLC or immunoassay (McNutt et al 2011; Yaguchi and Nishizaki 2010; Welch et al 2000), or indirect measurement detecting enzymatic breakdown products (McMahon and Nicholls 1991) or the postsynaptic currents by voltage clamping (Akaike et al 2010). The use of a fluorescent dye as a marker of neurotransmitter release has also been suggested (Rasetti-Escargueil et al 2011a; Tegenge et al 2012). Another potential but not yet developed endpoint is the measurement of electrical conductance of an entire population of cultured neurons.…”

Section: Neuronal Cell-based Assaysmentioning

confidence: 99%

Progress in Cell Based Assays for Botulinum Neurotoxin Detection

Pellett

2012

Current Topics in Microbiology and Immunology

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Botulinum neurotoxins (BoNTs) are the most potent human toxins known and the causative agent of botulism, and are widely used as valuable pharmaceuticals. The BoNTs are modular proteins consisting of a heavy chain and a light chain linked by a disulfide bond. Intoxication of neuronal cells by BoNTs is a multi-step process including specific cell binding, endocytosis, conformational change in the endosome, translocation of the enzymatic light chain into the cells cytosol, and SNARE target cleavage. The quantitative and reliable potency determination of fully functional BoNTs produced as active pharmaceutical ingredient (API) requires an assay that considers all steps in the intoxication pathway. The in vivo mouse bioassay has for years been the ‘gold standard’ assay used for this purpose, but it requires the use of large numbers of mice and thus causes associated costs and ethical concerns. Cell-based assays are currently the only in vitro alternative that detect fully functional BoNTs in a single assay and have been utilized for years for research purposes. Within the last 5 years, several cell-based BoNT detection assays have been developed that are able to quantitatively determine BoNT potency with similar or greater sensitivity than the mouse bioassay. These assays now offer an alternative method for BoNT potency determination. Such quantitative and reliable BoNT potency determination is a crucial step in basic research, in the development of pharmaceutical BoNTs, and in the quantitative detection of neutralizing antibodies.

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Enhanced sensitivity to Botulinum type A neurotoxin of human neuroblastoma SH-SY5Y cells after differentiation into mature neuronal cells

Cited by 11 publications

References 0 publications

Model for studying Clostridium botulinum neurotoxin using differentiated motor neuron-like NG108-15 cells

Model for studying Clostridium botulinum neurotoxin using differentiated motor neuron-like NG108-15 cells

High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics

Progress in Cell Based Assays for Botulinum Neurotoxin Detection

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