neurotoxicity assay using high content screening technology
TRANSCRIPT
Neurotoxicity assay using High Content Screening technology
N. Maubon1*, M. Roudaut1, N. Gueguen1, and J. Bursztyka1
1 HCS Pharma, Biopôle, 6 rue Pierre Joseph Colin, 35000 Rennes 1*[email protected]
Abstract
As shown by AstraZeneca in nature reviews*, one third of the safety failures is linked to CNS toxicity during the clinical trials of drugs . To avoid this attrition, the potential neurotoxicity of any drug going through the blood brain barrier (BBB) needs to be checked and if possible at the early stage of the research process for new chemical entites (NCE). This assay can be performed by cell imaging in HCA/HCS.
Methods
Results
Conclusions & Perspectives Neurotoxicity of compounds as rotenone or colchicine show that neurite length decreased in higher extent and/or at lower
concentration compared to death of neuronal cells. For example, neurite length decreased with colchicine at 0.01 µM while neuronal cell death appeared at 0.1 µM.
Some other compounds as Methylmercure show that neuronal cells are only impacted, showing high variability in neurite length. At high concentration of Methylmercure (at 100 µM for example), neurites already exist between neighbored resistant cells with high activity of mitochondria.
The last 2 other compounds (lead (PbCl2) and Valinomycine) showed toxicity in the same extent between neuronal cells and neurites. Conclusion : Neurotoxicity of compounds can be due to neuronal cell death but also due to loss of communication between the
neuronal cells and depending of neurotoxic compounds, the loss of communication (neurites) can appear at lowest dose than neuronal cell death. This model of neurotoxicity performed in HCS can be a good model during the early stage of research.
Cell culture: neuronal cells (SH-SY5Y) were routinely maintained in MEM/F12 (v/v) supplemented with 10% serum. Neuronal cells were seeded at 10 000 cells/well in 96-well Corning cellBind plates in MEM/F12 supplemented with differentiation agents. Then, cells were incubated at 37 °C in 5 % CO2 for 3 days for plating and differentiation.
Treatment assay : Neurotoxicity assay was performed by replaced medium in each well by fresh medium with the different compounds to be tested at different concentrations. After 48h of incubation, cells were incubated with mitotracker during 30 min, fixed and then stained with b III tubulin and Hoechst. Image acquisition was performed on Operetta plateform (Perkin Elmer) and the analysis of cell number and neurite length was done through Columbus software (Perkin Elmer).
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* Cook D, Brown D, Alexander R, March R, Morgan P, Satterthwaite G, Pangalos MN (2014) Lessons learned from the fate of AstraZeneca’s drug pipeline: a five-imensional framework. Nat Rev Drug Discov. 13(6):419-31.
CTRL Rotenone Colchicine Lead Me-mercure Valinomycine
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bIII-tubulin Hoechst
Mitotracker Hoechst
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