Parkinson's disease (PD), adult neurodegenerative disorder, is caused by the death of dopamine 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 death phenotype. Rotenone, a chemical compound commonly used as pesticide, is well-documented as death inducer of dopamine neurons in the substantia nigra and allow to mimic PD in vitro and in vivo. Rotenone-induced degeneration of dopaminergic neurons may not be solely attributed to an impairment of neuronal mitochondrial complex I activity in the dopaminergic neurons but may also be boosted by the participation of the resident immune cells in the brain: the microglia cells. Effectively, various environmental factors may also work in concert to induce dopaminergic neurodegeneration and numerous studies have confirmed that neuroinflammation plays a critical role in the pathogenesis of neurodegenerative deseases, including PD. To develop this assay, human neuroblastoma and mouse macrophagic cell lines in coculture were used, and rotenone was chosen as death inducer and phenotypic markers as Hoechst for nucleus and MAP2 for neurites analysis. Furthermore, first assays in 3D cell co-culture show promising results and give us nice perspectives for the future.

1. Development of co-culture Parkinson’s disease
model using High Content Screening technology
N. Maubon1, J. Bursztyka1, and V. Racine2
1nathalie.maubon@hcs-pharma.com ; 2victor.racine@quantacell.com
Abstract
Parkinson's disease (PD), adult neurodegenerative disorder, is caused by the death of dopamine 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 death phenotype. Rotenone,
a chemical compound commonly used as pesticide, is well-documented as death inducer of dopamine neurons in the substantia nigra and
allow to mimic PD in vitro and in vivo. Rotenone-induced degeneration of dopaminergic neurons may not be solely attributed to an
impairment of neuronal mitochondrial complex I activity in the dopaminergic neurons but may also be boosted by the participation of the
resident immune cells in the brain: the microglia cells. Effectively, various environmental factors may also work in concert to induce
dopaminergic neurodegeneration and numerous studies have confirmed that neuroinflammation plays a critical role in the pathogenesis of
neurodegenerative deseases, including PD.
To develop this assay, human neuroblastoma and mouse macrophagic cell lines in coculture were used, and rotenone was chosen as death
inducer and phenotypic markers as Hoechst for nucleus and MAP2 for neurites analysis.
Furthermore, first assays in 3D cell co-culture show promising results and give us nice perspectives for the future.
MethodsResults
Conclusions & Perspectives
 A nice dose toxicity effect of rotenone on coculture cells (neuronal and macrophages cells) was observed on cell death
and on neurite length with EC50 at around 0.2 µM for both.
 Neuroprotective effect on cell death induced by rotenone 1 µM in a context of neuroinflammation in high content
screening allows identifying new compounds and new pathways for PD treatment (assay was performed with K252a, ASK1
inhibitor: data not shown).
 First results on 3D cell culture on biomimesys® plates from CELENYS show that neuronal and macrophagic cells have two
different behaviors in culture: SH-SY5Y cells form spheroids while macrophagic cells stay separated.
 Perspective: This model will be developed in 3D culture when differentiation of neuronal cells in 3D culture will be
verified.
Cell culture: neuronal cells (SH-SY5Y) and macrophage cells (Raw264.7)
were routinely maintained in MEM/F12 (v/v) supplemented with 10%
serum. Neuronal cells were seeded alone or in co-culture with
macrophage cells 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 on 2D culture: Parkinson induction assay was performed
by replaced medium in each well by fresh medium with rotenone at
different concentrations with or without LPS. After 48h of incubation, cells
were fixed and stained with MAP2, and Hoechst. Image acquisition was
performed on ImPACcell platform (Arrayscan) and analysis was done
through Columbus (Perkin).
3D Cell culture: neuronal cells and macrophage cells were seeded alone or
in coculture at 25 000 cells/well in Biomimesys® plate from CELENYS in
MEM/F12 supplemented with differentiation agents. Then cells were
incubated at 37 °C in 5 % CO2 for 3 days for plating and differentiation.
Analysis and visualization were obtained using innovative algorithms and
software solutions dedicated to 3D cell culture. It allows the classification
of cell phenotypes and the measurement of 3D cell organization.
HoeschtMAP2
CTRL cells Rotenone (30 µM) treated cells
Rotenone treatment on 2D coculture
Rotenone treatment on 2D coculture3D co-culture of neuronal and macrophage cells
Neuronal cells Macrophage cells
Neuronal &
Macrophage cells
After 48H of incubation in Biomimesys® plates, magnitude: 40X
After 10 days of incubation in Biomimesys® plates, stained with Hoechst (blue) & MAP2 (green)
3D co-culture of neuronal and macrophage cells
Neuronal cells Macrophage cells
Neuronal &
Macrophage cells
rotenone CTRL
100 µM 30 µM 10 µM 3 µM 1 µM 0,3µM 0,1µM 0,03 µM 0,01 µM 0,003 µM
DMSO
0,5%
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