- Email: [email protected]
Development of functional cell-based assays as replacement assays for botulinum toxins and antitoxins
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Abstracts Toxins 2011 / Toxicon 68 (2013) 60–123
Development of functional cell-based assays as replacement assays for botulinum toxins and antitoxins C. Rasetti-Escargueil a, C.B. Machado b, P. Charlesworth c, L. Kolker a, R.A. Fleck a, D. Sesardic a
multi-electrode-arrays (MEAs) provide a suitable basis for the development of a cell-based assay of botulinum toxin action on neuronal networks. http://dx.doi.org/10.1016/j.toxicon.2012.07.104
a
National Institute for Biological Standards and Control, Health Protection Agency,Potters Bar, UK b MRC Centre for Developmental Neurobiology, King's College andGuy's Campus, London, UK c Department of Physiology, Development and Neuroscience Physiological Laboratory, Cambridge University,Cambridge, UK E-mail address: [email protected] (D. Sesardic).
In vivo functional actions of type A botulinum toxins M.C. Shin a, N. Akaike a, K. Nonaka a, M. Wakita a, Y. Torii b, T. Harakawa b, A. Ginnaga b a b
Purpose of study: Botulinum neurotoxins are lethal toxins that induce prolonged muscle paralysis by blocking the release of neuronal transmitters from peripheral cholinergic nerve endings. Although highly toxic, botulinum toxins are available as licensed drugs for the treatment of a variety of medical disorders and increasingly applied for cosmetic purposes, which results in substantial increase of animal use in the lethality assays worldwide. Recent review of existing alternative methods by international experts recommended research on cellular neurotoxicity mechanisms for novel test assays as essential for progress. In this study application of the multi-electrodearray (MEAs) platform as a potential functional neurotoxicity assay was implemented in parallel with vesicle trafficking and neurotransmitter release measurements. Investigating the effects of botulinum toxin on neuronal communication will reveal fresh insights needed to revive cell-based assay development and to develop improved therapeutic countermeasures. Methods used: Human neuroblastoma cell and murine embryonic stem cell lines were established in culture and differentiated into mature neuronal cells. Vesicle turnover was explored after exposure to a wide range of concentrations of type A botulinum toxin in parallel with radiolabelled neurotransmitter measurements. Non-invasive recordings of spontaneous electrical activities were performed on neuronal networks cultured on PEI and on laminin-coated MEA dishes embedded with a square array of 64 microelectrodes. Summary of results: Accurate vesicle turnover assessments were obtained using flow cytometry, which were dose-dependently inhibited after exposure to botulinum toxin. Uptake of FM1-43 was markedly reduced in cultures treated with botulinum type A toxin and partly restored in cultures exposed to toxin previously incubated with type A antitoxin.Reproducible and robust differentiation protocols generated stable neuronal cultures that formed extensive networks over the multi-electrode culture dish surface. Differentiated SH-SY5Y cells and murine stem cells-derived neuronal networks exhibited spontaneous activity until up to six weeks after seedingonto MEAs,indicating that networks of cultured neuronal cells represent a potential model system for studying botulinum toxin neurotoxicity. Conclusions: Overall, these findings indicate that a fully differentiated human cell line and embryonic stem cells on
Kumamoto Health Science University, Kumamoto, Japan Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan E-mail address: [email protected] (K. Nonaka).
Purpose of study: Botulinum toxin is one of the most toxic substances in nature. However, when injected therapeutically in small doses, the toxin is a safe and effective treatment for a variety of medical disorders. We have attempted to produce neurotoxin from subtype A2 (A2NTX). The molecular size is much smaller (150 kDa) than that of A1 type botulinum toxin (A1LL, 900 kDa). Here we examined how differently A1LL and A2NTX modulate behavioral activities and muscle contraction under in vivo conditions. Methods used: To investigate the effect of A1LL and A2NTX on treadmill running time under in vivo conditions, adult rats were injected with toxins into the soleus muscle, and running time over five days after the toxin injection was measured. Also, to investigate muscle flaccid effect of A1LL and A2NTX, these neurotoxins were injected into soleus muscle of one leg side, andthe ipsi- and contralateral muscle contraction was measured over five days after injection. Summary of results: In the treadmill study A2NTX suppressed treadmill running time in a concentrationdependent fashion, and the inhibitory effect of 12 U/kg A2NTX was potent and faster than that of 12 U A1LL. In the colchicine- and antitoxin-treated rat models, A2NTX completely blocked the twitch contraction evoked by indirect nerve stimulations of the toxin- injected ipsilateral soleus muscle without affecting the direct muscle stimulation. A2NTX of high doses (12 and 40 U) affected the contralateral soleus muscles in a concentrationdependent fashion via either axonal flow or blood. Similar toxin diffusion to the contralateral muscle was also observed after A1LL injection, where A1LL was mainly transferred to contralateral muscles by axonal flow. Conclusions: It was confirmed that A2NTX is a more potent and faster-acting muscle relaxant than A1LL. On toxin diffusion pathway from toxin-injected ipsilateral muscle to the contralateral one, A2NTX and A1LL are mainly transported through blood and nerve, respectively. These results suggest that A2NTX is used as a therapeutic agent for neurological disorders with somewhat different way from A1LL. http://dx.doi.org/10.1016/j.toxicon.2012.07.105
Abstracts Toxins 2011 / Toxicon 68 (2013) 60–123
Development of functional cell-based assays as replacement assays for botulinum toxins and antitoxins C. Rasetti-Escargueil a, C.B. Machado b, P. Charlesworth c, L. Kolker a, R.A. Fleck a, D. Sesardic a
multi-electrode-arrays (MEAs) provide a suitable basis for the development of a cell-based assay of botulinum toxin action on neuronal networks. http://dx.doi.org/10.1016/j.toxicon.2012.07.104
a
National Institute for Biological Standards and Control, Health Protection Agency,Potters Bar, UK b MRC Centre for Developmental Neurobiology, King's College andGuy's Campus, London, UK c Department of Physiology, Development and Neuroscience Physiological Laboratory, Cambridge University,Cambridge, UK E-mail address: [email protected] (D. Sesardic).
In vivo functional actions of type A botulinum toxins M.C. Shin a, N. Akaike a, K. Nonaka a, M. Wakita a, Y. Torii b, T. Harakawa b, A. Ginnaga b a b
Purpose of study: Botulinum neurotoxins are lethal toxins that induce prolonged muscle paralysis by blocking the release of neuronal transmitters from peripheral cholinergic nerve endings. Although highly toxic, botulinum toxins are available as licensed drugs for the treatment of a variety of medical disorders and increasingly applied for cosmetic purposes, which results in substantial increase of animal use in the lethality assays worldwide. Recent review of existing alternative methods by international experts recommended research on cellular neurotoxicity mechanisms for novel test assays as essential for progress. In this study application of the multi-electrodearray (MEAs) platform as a potential functional neurotoxicity assay was implemented in parallel with vesicle trafficking and neurotransmitter release measurements. Investigating the effects of botulinum toxin on neuronal communication will reveal fresh insights needed to revive cell-based assay development and to develop improved therapeutic countermeasures. Methods used: Human neuroblastoma cell and murine embryonic stem cell lines were established in culture and differentiated into mature neuronal cells. Vesicle turnover was explored after exposure to a wide range of concentrations of type A botulinum toxin in parallel with radiolabelled neurotransmitter measurements. Non-invasive recordings of spontaneous electrical activities were performed on neuronal networks cultured on PEI and on laminin-coated MEA dishes embedded with a square array of 64 microelectrodes. Summary of results: Accurate vesicle turnover assessments were obtained using flow cytometry, which were dose-dependently inhibited after exposure to botulinum toxin. Uptake of FM1-43 was markedly reduced in cultures treated with botulinum type A toxin and partly restored in cultures exposed to toxin previously incubated with type A antitoxin.Reproducible and robust differentiation protocols generated stable neuronal cultures that formed extensive networks over the multi-electrode culture dish surface. Differentiated SH-SY5Y cells and murine stem cells-derived neuronal networks exhibited spontaneous activity until up to six weeks after seedingonto MEAs,indicating that networks of cultured neuronal cells represent a potential model system for studying botulinum toxin neurotoxicity. Conclusions: Overall, these findings indicate that a fully differentiated human cell line and embryonic stem cells on
Kumamoto Health Science University, Kumamoto, Japan Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan E-mail address: [email protected] (K. Nonaka).
Purpose of study: Botulinum toxin is one of the most toxic substances in nature. However, when injected therapeutically in small doses, the toxin is a safe and effective treatment for a variety of medical disorders. We have attempted to produce neurotoxin from subtype A2 (A2NTX). The molecular size is much smaller (150 kDa) than that of A1 type botulinum toxin (A1LL, 900 kDa). Here we examined how differently A1LL and A2NTX modulate behavioral activities and muscle contraction under in vivo conditions. Methods used: To investigate the effect of A1LL and A2NTX on treadmill running time under in vivo conditions, adult rats were injected with toxins into the soleus muscle, and running time over five days after the toxin injection was measured. Also, to investigate muscle flaccid effect of A1LL and A2NTX, these neurotoxins were injected into soleus muscle of one leg side, andthe ipsi- and contralateral muscle contraction was measured over five days after injection. Summary of results: In the treadmill study A2NTX suppressed treadmill running time in a concentrationdependent fashion, and the inhibitory effect of 12 U/kg A2NTX was potent and faster than that of 12 U A1LL. In the colchicine- and antitoxin-treated rat models, A2NTX completely blocked the twitch contraction evoked by indirect nerve stimulations of the toxin- injected ipsilateral soleus muscle without affecting the direct muscle stimulation. A2NTX of high doses (12 and 40 U) affected the contralateral soleus muscles in a concentrationdependent fashion via either axonal flow or blood. Similar toxin diffusion to the contralateral muscle was also observed after A1LL injection, where A1LL was mainly transferred to contralateral muscles by axonal flow. Conclusions: It was confirmed that A2NTX is a more potent and faster-acting muscle relaxant than A1LL. On toxin diffusion pathway from toxin-injected ipsilateral muscle to the contralateral one, A2NTX and A1LL are mainly transported through blood and nerve, respectively. These results suggest that A2NTX is used as a therapeutic agent for neurological disorders with somewhat different way from A1LL. http://dx.doi.org/10.1016/j.toxicon.2012.07.105