HAL Id: hal-02748799
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Submitted on 3 Jun 2020
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Effect of uranium on multipotency of neural stem cells in a primary neurosphere culture model
Antoine Becquet, Celine Gloaguen, Karine Tack, Chrystelle Ibanez
To cite this version:
Antoine Becquet, Celine Gloaguen, Karine Tack, Chrystelle Ibanez. Effect of uranium on multipotency of neural stem cells in a primary neurosphere culture model. 55th Congress of the European Societies of Toxicology, EUROTOX, Sep 2019, HELSINKI, Finland. �hal-02748799�
DISCUSSION RESULTS
AIMS AND EXPERIMENTAL PROTOCOL INTRODUCTION
Effect of uranium on multipotency of neural stem cells in a primary neurosphere culture model
Antoine Becquet, Céline Gloaguen, Karine Tack, Chrystelle Ibanez
Institut de Radioprotection et de Sûreté Nucléaire, Pôle Santé Environnement, Service de recherche sur les effets biologiques et sanitaires des rayonnements ionisants, Laboratoire de Radiotoxicologie et Radiobiologie Expérimentale, BP17, 92262 Fontenay aux Roses Cedex, France.
Uranium exposure situations are diverse and originate from its natural presence in the environment, and from its use in specific professional activities in relation with the nuclear industry (extraction, nuclear fuel cycles, and dismantling operations).
Uranium internal contamination can occur via ingestion of contaminated food and drinking water or via inhalation of particulate aerosols containing uranium dust. This latest situation is the main cause of contamination in nuclear occupational activities. These contaminations raise concern in terms of potential consequences on human health. They appear to have negative impact on the brain as experimental studies have shown that uranium exposure via ingestion or inhalation can lead to cognitive impairments in rats. Neurogenesis disruption has been proposed to underlie these effects. To address this question, we used in vitro neurosphere primary cultures from rat embryo’s telencephalon at embryonic day 13. We studied uranium impact on multipotency of neural stem cell within a range of concentrations (10, 50, 100 µM) versus control over 7 days of contamination. Results and discussion are provided below.
These results suggest that uranium exerts a specific action on late cell maturation phases rather than on early determination stages.
55th Congress of the European Societies of Toxicology – 8-11 September, 2019 – Helsinki, Finland
RT-qPCR
Glial progenitors and mature oligodendrocytes Neurons and astrocytes
Neuron morphology
A
▌ In vitro model : neurosphere primary cultures
▌ Explore mechanisms of action
Multipotency : target phases ? Neurogenesis + gliogenesis ?
mRNA expression of neurogenic and apoptotic markers
▌ Identify target cells
Neural stem cells, progenitors, mature cells:
neurons, astrocytes, oligodendrocytes
▌
Uranium is a radioactive heavy metal
Chemical toxicity
Radiotoxicity
▌ Neuronal morphology alteration after 7 days of contamination
Reduction of axonal length
Dendrites atrophy
0.0%
20.0%
40.0%
60.0%
80.0%
0 20 40 60 80
Témoin 10µM 50µM 100µM
Cell number
Astrocyte population (GFAP+) and proportion
*
*
*
0.0%
10.0%
20.0%
30.0%
0 10 20 30
Témoin 10µM 50µM 100µM
Cell Number
Neuron population (βTub+) and proportion
* *
# #
#
0.0%
20.0%
40.0%
60.0%
80.0%
0 40 80 120 160
Témoin 10µM 50µM 100µM
Cell number
Glial cell lineage population (Olig2+) and proportion
*
###
0.0%
2.0%
4.0%
6.0%
8.0%
0 5 10 15 20
Témoin 10µM 50µM 100µM
Cell number
Oligodendrocytes population (MBP+) and proportion
*
*
# #
Neural stem cell differentiation
GLIOGENESIS NEUROGENESIS
Oligodendrocyte Astrocyte Neuron
MATURATION
DIFFERENTIATION
SELF RENEWAL
DETERMINATION
Neural progenitor:
Multipotent Symmetric division Rapid cell cycle
Neural stem cell:
Multipotent Asymmetric division Slow cell cycle
or or
Neuronal progenitor Astrocyte
progenitor Oligodendrocyte
progenitor
▌ Significant reduction of neuron number from 50 µM
▌ Significant decrease of neuron proportion from 10 µM
▌ Significant reduction of astrocyte number from 50 µM
▌ Significant increase of the proportion of Olig2+
progenitors at 100 µM in parallel with a significant decrease of cell number
▌ Significant decrease of the number and proportion of mature oligodendrocyte from 50 µM
-0.50 0.00 0.50 1.00 1.50 2.00 2.50 3.00
10µM 50µM 100µM
Apoptotic marker expression
Bcl2 Bax Caspase3 Caspase9
*
0.00 1.00 2.00 3.00 4.00 5.00
10µM 50µM 100µM
Neurogenic marker expression
Mash1 (ASCL1) Notch1 NeuroD NeuroG3 GAP43
Determination Differentiation Maturation
**
*
▌
Significant decrease of caspase 9 mRNA expression at 10 µM (effector of pro- apoptotic caspase 3)
▌
Switch of bcl2/Bax balance at 100 µM towards an anti-apoptotic profile
▌
Hypothesis : set-up of an adaptative mechanism of surviving cells after 7 days of contamination ?
▌
Neuronal atrophy at 50 and 100 µM
▌
Significant decrease of GAP43 mRNA expression at 100 µM
GAP43 is involved in the development of neuron dendrites
▌
Hypothesis : Impairment of the terminal neuron maturation phase leading to impaired synaptic
contacts ?
▌
Significant increase of the proportion of glial progenitors
▌
Significant decrease of the proportion of mature oligodendrocytes
▌
Significant increase of NeuroG3 mRNA expression at 100 µM
▌
Hypothesis : cells remain in a non mature phase of differentiation due to NeuroG3 mRNA overexpression ?
▌
Cells from the oligodendrocyte
lineage appear to be more sensitive to uranium compared to the
astrocytic lineage ?
GLIOGENESIS NEUROGENESIS
Oligodendrocyte Astrocyte Neuron
MATURATION
DIFFERENTIATION
SELF RENEWAL
DETERMINATION
Neural progenitor:
Multipotent Symmetric division Rapid cell cycle
Neural stem cell:
Multipotent Asymmetric division Slow cell cycle
or or
Neuronal progenitor Astrocyte
progenitor Oligodendrocyte
progenitor
▌ Differentiated neural stem cells after 7 days of contamination (control, 10, 50, 100 µM)
Acknowledgements for illustrations by Charline Pichot (Histogram: absolute number, Line: proportion)
(Histogram: absolute number, Line: proportion) Control
MEMBER OF
Control Control Control
Control
E13 embryo
Telencephalon
10µM T 50µM 100µM
Proliferation
Differentiation
EGF
bFGF + SVF
+ DU*
*DU: depleted uranium UO2(NO3)2,6H2O
7 jours
7 days
10µM T 50µM 100µM
Immunostaining
&
Quantification
RT-qPCR
