AbstractParkinson's disease (PD), a neurodegenerative disorder, is caused by the death of dopaminergic neurons in the substantia nigra. High contentscreening (HCS) should allow finding new pathways involved in the onset of PD by screening molecules based on phenotypes related to celldeath. Rotenone, a chemical compound commonly used as a pesticide, is well-documented as a cell death inducer of dopaminergic neuronsin 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 thisneuroprotection 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 3Dculture 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 allowsidentifying 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 neuritesinduction by a differentiation agent. Neurites are present on the surface of spheroids but not inside. They connect differentspheroids and spheroids with isolated cells.

Perspective: This model in 3D should be more relevant to observe specifically modification in neurite connection but more analysisneed 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 000cells/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 daysfor 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 ornot with amantadine at different concentrations. After 48h of incubation, cells were fixed and stained with b3-tubulin (green) and Hoechst (blue). Imageacquisition 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 cellswere 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

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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 Rennes2 Perkin Elmer , 16 av Québec, Bât Lys, 91140 Villebon sur Yvette

1*nathalie.maubon@hcs-pharma.com