Parkinson's disease (PD), a neurodegenerative disorder, is caused by the death of dopaminergic neurons in the substantia nigra. High content screening (HCS) should allow finding new pathways involved in the onset of PD by screening molecules based on phenotypes related to cell death. Rotenone, a chemical compound commonly used as a pesticide, is well-documented as a cell death inducer of dopaminergic neurons in the substantia nigra and allows mimicking PD in vitro and in vivo. HCS Pharma is working to develop new, more complex and relevant in vitro cellular models that mimic the pathology as closely as possible. In this poster, a neuroprotection model of PD is presented, with differenciated SH-SY5Y neuronal cells, exposed to rotenone. Model was developed in 2D culture and first results in 3D culture are also presented.

1. Abstract
Parkinson's disease (PD), a neurodegenerative disorder, is caused by the death of dopaminergic neurons in the substantia nigra. High content
screening (HCS) should allow finding new pathways involved in the onset of PD by screening molecules based on phenotypes related to cell
death. Rotenone, a chemical compound commonly used as a pesticide, is well-documented as a cell death inducer of dopaminergic neurons
in the substantia nigra and allows mimicking PD in vitro and in vivo.
Amantadine is commonly given to alleviate L-DOPA-induced dyskinesia of Parkinson’s disease (PD) patients. It is generally believed that this
neuroprotection results from the ability of amantadine to inhibit glutamatergic NMDA receptor.
In this poster, a neuroprotection model of PD is presented, with differenciated SH-SY5Y neuronal cells, exposed to rotenone. In this assay,
amantadine showed a significatively effect of neuroprotection at 10 and 30 nM. This model was developed in 2D culture and first results in 3D
culture are also presented.
Methods
Results
Conclusions & Perspectives
 In a previous study, a nice dose toxicity effect of rotenone on differentiated SH-SY5Y cells was observed on cell death and on neurite
length with EC50 at around 0.2 µM for both.
 In this study, neuroprotective effect of amantadine 10 and 30 nM, a well known Parkinson drug treatment, was observed on cell
death induced by rotenone 1 µM => neuroprotective effect on cell death induced by rotenone 1 µM in high content screening allows
identifying new compounds and new pathways for PD treatment.
 First results on 3D cell culture show that neuronal cells cultured in spheroids can connect spheroids between them with neurites
induction by a differentiation agent. Neurites are present on the surface of spheroids but not inside. They connect different
spheroids and spheroids with isolated cells.
 Perspective: This model in 3D should be more relevant to observe specifically modification in neurite connection but more analysis
need to be performed to develop this assay in 3D.
Cell culture: neuronal cells (SH-SY5Y) were routinely maintained in MEM/F12 (v/v) supplemented with 10% FBS. Neuronal cells were seeded alone at 10 000
cells/well in 96-well Corning cellbind plates in MEM/F12 supplemented with a differentiation agent. Then, cells were incubated at 37 °C in 5 % CO2 for 3 days
for plating and differentiation.
Treatment assay on 2D culture: Parkinson induction assay was performed by replaced medium in each well by fresh medium with rotenone 1 µM co-treated or
not with amantadine at different concentrations. After 48h of incubation, cells were fixed and stained with b3-tubulin (green) and Hoechst (blue). Image
acquisition was performed on Operetta and analysis was done through Columbus (PerkinElmer).
3D Cell culture: neuronal cells were seeded at 100 000 cells/well on medium binding plate in MEM/F12 supplemented with a differentiation agent. Then cells
were incubated at 37°C in 5% CO2 for 3 days for plating and differentiation. Analysis and visualization were performed on Columbus and Volocity
(PerkinElmer).
CTRL cells Rotenone 1 µM treated cells
Neuroprotection effect of amantadine against SH-SY5Y cell
death induced by rotenone on 2D culture
3D culture of SH-SY5Y neuronal cells in presence with
differentiation agent
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Cotreatment of amantadine 30 nM and
rotenone 1 µM
Dose effect of amantadine cotreated with
rotenone 1 µM
(Live cells measurement)
Maximum projection Retreated picture for analysis
Different Z planes from bottom
(1st picture) to top (last picture) of
the spheroid
Z-cut visualization of the spheroid : Green
staining is observed only of the surface of
the spheroid, not inside.
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CTRL rotenone 1 µM + Amantadine
**
* p<0.05
Development of a neuroprotection assay for Parkinson’s
disease in vitro model using High Content Screening
MAUBON N.1*, ROUDAUT M.1, NDOYE A.2 & BURSZTYKA J.1
1 HCS Pharma, 6 rue Pierre Joseph Colin, 35000 Rennes
2 Perkin Elmer , 16 av Québec, Bât Lys, 91140 Villebon sur Yvette
1*nathalie.maubon@hcs-pharma.com