Effects of pesticides on autophagic flux and cell death

ECOLE DOCTORALE "Médicament, Toxicologie, Chimie, Imageries"
- UNIVERSITE PARIS DESCARTES
Proposition de sujet de thèse à l’appui d’une demande de contrat doctoral 2014-2015
Renseignements relatifs à l’Unité de Recherche :
Nom et appartenance : INSERM U 1124
Nom et prénom du Directeur : Prof. Robert BAROUKI
Téléphone : 33 (0)1 42 86 20 75
Courriel : [email protected]
Signature obligatoire :(et avis éventuel) :
Renseignements relatifs à l’Equipe d’Accueil (EAD) :
Nom de l’Equipe d’Accueil : Equipe 1
Nom et prénom du responsable : Prof. Xavier COUMOUL
Qualité du responsable : Professeur
Téléphone : 33 (0)1 42 86 33 59
courriel : [email protected]
Signature obligatoire:
Renseignements relatifs au sujet de thèse :
Nom et prénom du Directeur de thèse (Docteur d’Etat es Sciences) : Dr. Patrice X. PETIT
Qualité : Directeur de Recherche CNRS (DR2)
Téléphone : 33 (0)1 42 86 20 73
courriel : [email protected]
Signature obligatoire :
secteurs disciplinaires (510 principal et 530 secondaire)
Titre du sujet proposé :
Effets de pesticides sur la modulation des flux autophagiques et la mort cellulaire :
Implication dans le cadre de la maladie de Parkinson et des lymphomes non Hodgkiniens
Un groupe d’expert INSERM vient de remettre un rapport concernant entre autres choses, les
liens avérés ou soupçonnés entre pesticides et de multiples pathologies dont les lymphomes non
Hodgkiniens et la maladie de Parkinson. Les pesticides modifient le métabolisme cellulaire et la
bioénergétique mitochondriale induisant de multiples perturbations susceptibles d’affecter les
mécanismes autophagiques et de provoquer ou non la mort cellulaire. En regard de notre expertise,
nous allons aborder l’étude du statut bioénergétique des cellules, des flux autophagiques et des
mécanismes de mort associés à l’aide d’un ensemble de techniques d’imagerie, de cytométrie en flux et
de biochimie classique. Ces mécanismes seront étudiés sur des lymphomes non Hodgkiniens et sur des
modèles de la maladie de Parkinson.
Demande dans le cadre d’un projet ARC 2014 (dépôt prévu - 2ème trimestre 2014)
ECOLE DOCTORALE "Médicament, Toxicologie, Chimie, Imageries"
- UNIVERSITE PARIS DESCARTES
Proposition de sujet de thèse à l’appui d’une demande de contrat doctoral 2014-2015
Nom, prénom du directeur de l'unité de recherche : Prof. Robert BAROUKI
Numéro de l'unité de recherche (et établissement de rattachement) : U1124 (Université Paris-Descartes)
Nom, prénom du responsable de l'équipe d'accueil (EAD) : Prof. Xavier COUMOUL
Nom, prénom du directeur de thèse : Dr. Patrice X. PETIT
Effects of pesticides on autophagic flux and cell death :
Implications on Parkinson disease model and non-Hodgkin lymphoma.
A recent report of group expert at INSERM highlighted the relationship between various
pesticides, non-hodgkin lymphoma and Parkinson disease. Since our team has an extended expertise
on cancer cells treated with a mixture of persistent organic pollutants (POPs), we will extend this area of
expertise with these two models of choice to pinpoint interactions between pesticides and the cellular
metabolism in a pathologic context. Our work will take a more fundamental aspect since the pesticides
will not only be used to depict their original mode of action but also as tools to precise signal transduction
pathways. That include destabilization of the mitochondrial compartment together with the ER and the
lysosomes, in a context where a special attention with be taken to investigate the modulation of the
autophagic flux and the aspects of the so called «autophagic death» if any.
Current results demonstrate that diverse pepticides not only destabilize the cellular metabolism,
but also trigger mitochondrial bioenergetic changes, ROS production and calcium rise. All together,
these events may affect constitutive autophagic flux and cellular sensitivity to death. Products such as
rotenone (the prototypic inducer of Parkinson), 1-methyl-4-phenylpyridinium (Parkinson mimetic) are
usually used to modelize Parkinson disease associated events, but also endosulfan (POPs), glyphosate
(most worldwidely used soluble pesticide) and 6-hydroxydopamine (neurotoxin) will be tested for their
relationship to Parkinson.
Autophagy is an evolutionarily conserved subcellular degradation process engulfing proteins,
proteins complexes, misfolded proteins aggregates and entire organelles such as damaged
mitochondria. Classically contributing to cellular homeostasis and adaptation to stress, the autophagic
machinery is now recognized to be in motion during several pathogenic conditions and diseases, and for
the execution of a caspase-independant cell death. It is clear that pesticides may change autophagic flux
as the main signal inducing autophagy is mediated through ROS.
To study those mechanisms we'll benefit from different cell lines, both normal and malignant (see
below: cell lines) and consider current test available: Classic qPCR and Western blot to test LC3B,
Beclin-1, p62, caspase-3/-7/-8/-9, calpain, cathepsin as well as Bcl-2, Bax and/or Bak; LC3-GFP and
GFP-RFP-LC3B construction for confocal microscopy; Metabolic activities by flow cytometry
(integrity/viability, ΔΨm measurements, superoxide anion and hydroperoxide production) and cell cycle.
Oxygen measurements throughout Seahorse or Oroboros equipments to assess oxidative properties of
pepticides treated cells.
We want to go further by assessing hypotheses that ROS drive a part of pesticides effects
(essentially with glyphosate), subsequently change calcium flux, weighing directly or indirectly on
Mitochondrial calcium uniporter (MCU). Standing on recent discovery of MCU and regulatory subunits,
the use of microRNA technology (miR25), involvement of ER for calcium release, and lysosomes for
autophagy (miR95) (Innovative targeted nanoparticles allowing us to restore their functions if altered) we
can draw a global mechanistic response to pesticides.
We are eager to enlighten relationship between pesticides, radical oxygen production, calcium
influx and cancer agressiveness on one side and Parkinson disease on the other side relying on the
interplay between autophagy and subsequent death mechanisms as the latter fail to protect cells.
Cell lines: 1C11 cells (can be differentiated in dopaminergic or serotoninergic neurons, provided by Benoit
Schneider), N27 cells, SH-SY5Ycells or PC12 cells.
For the Non Hodgkinian lymphoma : P3HR1, Ramos or Raji cells lines (lymphome B).

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