Development of a stretch-induced neurotrauma model for medium-throughput screening in vitro

Identification of rifampicin as a neuroprotectant

Isabel López-García, Domokos Gero, Bartosz Szczesny, Petra Szoleczky, Gabor Olah, Katalin Modis, Kangling Zhang, Junling Gao, Ping Wu, Lawrence Sowers, Douglas Dewitt, Donald Prough, Csaba Szabo

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Abstract

Background and Purpose: We hypothesized that an in vitro, stretch-based model of neural injury may be useful to identify compounds that decrease the cellular damage in neurotrauma. Experimental Approach: We screened three neural cell lines (B35, RN33B and SH-SY5Y) subjected to two differentiation methods and selected all-trans-retinoic acid-differentiated B35 rat neuroblastoma cells subjected to rapid stretch injury, coupled with a subthreshold concentration of H2O2, for the screen. The model induced marked alterations in gene expression and proteomic signature of the cells and culminated in delayed cell death (LDH release) and mitochondrial dysfunction [reduced 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) conversion]. Follow-up studies utilized human stem cell-derived neurons subjected to rapid stretch injury. Key Results: From screening of a composite library of 3500 drugs, five drugs (when applied in a post-treatment regimen relative to stretch injury) improved both LDH and MTT responses. The effects of rifampicin were investigated in further detail. Rifampicin reduced cell necrosis and apoptosis and improved cellular bioenergetics. In a second model (stretch injury in human stem cell-derived neurons), rifampicin pretreatment attenuated LDH release, protected against the loss of neurite length and maintained neuron-specific class III β-tubulin immunoreactivity. Conclusions and Implications: We conclude that the current model is suitable for medium-throughput screening to identify compounds with neuroprotective potential. Rifampicin, when applied either in pre- or post-treatment, improves the viability of neurons subjected to stretch injury and protects against neurite loss. Rifampicin may be a candidate for repurposing for the therapy of traumatic brain injury.

Original languageEnglish (US)
JournalBritish Journal of Pharmacology
DOIs
StateAccepted/In press - 2016

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Neuroprotective Agents
Rifampin
Wounds and Injuries
Neurons
Neurites
Stem Cells
Tubulin
Tretinoin
Neuroblastoma
Transcriptome
Pharmaceutical Preparations
Proteomics
Energy Metabolism
In Vitro Techniques
Cell Death
Necrosis
Apoptosis
Cell Line

ASJC Scopus subject areas

  • Pharmacology

Cite this

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title = "Development of a stretch-induced neurotrauma model for medium-throughput screening in vitro: Identification of rifampicin as a neuroprotectant",
abstract = "Background and Purpose: We hypothesized that an in vitro, stretch-based model of neural injury may be useful to identify compounds that decrease the cellular damage in neurotrauma. Experimental Approach: We screened three neural cell lines (B35, RN33B and SH-SY5Y) subjected to two differentiation methods and selected all-trans-retinoic acid-differentiated B35 rat neuroblastoma cells subjected to rapid stretch injury, coupled with a subthreshold concentration of H2O2, for the screen. The model induced marked alterations in gene expression and proteomic signature of the cells and culminated in delayed cell death (LDH release) and mitochondrial dysfunction [reduced 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) conversion]. Follow-up studies utilized human stem cell-derived neurons subjected to rapid stretch injury. Key Results: From screening of a composite library of 3500 drugs, five drugs (when applied in a post-treatment regimen relative to stretch injury) improved both LDH and MTT responses. The effects of rifampicin were investigated in further detail. Rifampicin reduced cell necrosis and apoptosis and improved cellular bioenergetics. In a second model (stretch injury in human stem cell-derived neurons), rifampicin pretreatment attenuated LDH release, protected against the loss of neurite length and maintained neuron-specific class III β-tubulin immunoreactivity. Conclusions and Implications: We conclude that the current model is suitable for medium-throughput screening to identify compounds with neuroprotective potential. Rifampicin, when applied either in pre- or post-treatment, improves the viability of neurons subjected to stretch injury and protects against neurite loss. Rifampicin may be a candidate for repurposing for the therapy of traumatic brain injury.",
author = "Isabel L{\'o}pez-Garc{\'i}a and Domokos Gero and Bartosz Szczesny and Petra Szoleczky and Gabor Olah and Katalin Modis and Kangling Zhang and Junling Gao and Ping Wu and Lawrence Sowers and Douglas Dewitt and Donald Prough and Csaba Szabo",
year = "2016",
doi = "10.1111/bph.13642",
language = "English (US)",
journal = "British Journal of Pharmacology",
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TY - JOUR

T1 - Development of a stretch-induced neurotrauma model for medium-throughput screening in vitro

T2 - Identification of rifampicin as a neuroprotectant

AU - López-García, Isabel

AU - Gero, Domokos

AU - Szczesny, Bartosz

AU - Szoleczky, Petra

AU - Olah, Gabor

AU - Modis, Katalin

AU - Zhang, Kangling

AU - Gao, Junling

AU - Wu, Ping

AU - Sowers, Lawrence

AU - Dewitt, Douglas

AU - Prough, Donald

AU - Szabo, Csaba

PY - 2016

Y1 - 2016

N2 - Background and Purpose: We hypothesized that an in vitro, stretch-based model of neural injury may be useful to identify compounds that decrease the cellular damage in neurotrauma. Experimental Approach: We screened three neural cell lines (B35, RN33B and SH-SY5Y) subjected to two differentiation methods and selected all-trans-retinoic acid-differentiated B35 rat neuroblastoma cells subjected to rapid stretch injury, coupled with a subthreshold concentration of H2O2, for the screen. The model induced marked alterations in gene expression and proteomic signature of the cells and culminated in delayed cell death (LDH release) and mitochondrial dysfunction [reduced 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) conversion]. Follow-up studies utilized human stem cell-derived neurons subjected to rapid stretch injury. Key Results: From screening of a composite library of 3500 drugs, five drugs (when applied in a post-treatment regimen relative to stretch injury) improved both LDH and MTT responses. The effects of rifampicin were investigated in further detail. Rifampicin reduced cell necrosis and apoptosis and improved cellular bioenergetics. In a second model (stretch injury in human stem cell-derived neurons), rifampicin pretreatment attenuated LDH release, protected against the loss of neurite length and maintained neuron-specific class III β-tubulin immunoreactivity. Conclusions and Implications: We conclude that the current model is suitable for medium-throughput screening to identify compounds with neuroprotective potential. Rifampicin, when applied either in pre- or post-treatment, improves the viability of neurons subjected to stretch injury and protects against neurite loss. Rifampicin may be a candidate for repurposing for the therapy of traumatic brain injury.

AB - Background and Purpose: We hypothesized that an in vitro, stretch-based model of neural injury may be useful to identify compounds that decrease the cellular damage in neurotrauma. Experimental Approach: We screened three neural cell lines (B35, RN33B and SH-SY5Y) subjected to two differentiation methods and selected all-trans-retinoic acid-differentiated B35 rat neuroblastoma cells subjected to rapid stretch injury, coupled with a subthreshold concentration of H2O2, for the screen. The model induced marked alterations in gene expression and proteomic signature of the cells and culminated in delayed cell death (LDH release) and mitochondrial dysfunction [reduced 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) conversion]. Follow-up studies utilized human stem cell-derived neurons subjected to rapid stretch injury. Key Results: From screening of a composite library of 3500 drugs, five drugs (when applied in a post-treatment regimen relative to stretch injury) improved both LDH and MTT responses. The effects of rifampicin were investigated in further detail. Rifampicin reduced cell necrosis and apoptosis and improved cellular bioenergetics. In a second model (stretch injury in human stem cell-derived neurons), rifampicin pretreatment attenuated LDH release, protected against the loss of neurite length and maintained neuron-specific class III β-tubulin immunoreactivity. Conclusions and Implications: We conclude that the current model is suitable for medium-throughput screening to identify compounds with neuroprotective potential. Rifampicin, when applied either in pre- or post-treatment, improves the viability of neurons subjected to stretch injury and protects against neurite loss. Rifampicin may be a candidate for repurposing for the therapy of traumatic brain injury.

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U2 - 10.1111/bph.13642

DO - 10.1111/bph.13642

M3 - Article

JO - British Journal of Pharmacology

JF - British Journal of Pharmacology

SN - 0007-1188

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