1. In Vivo Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae
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Baptiste Grimaud, Maxence Frétaud, Feriel Terras, Antoine Bénassy, Karine Duroure, Valérie Bercier, Gaëlle Trippé-Allard, Rabei Mohammedi, Thierry Gacoin, Filippo Del Bene, François Marquier, Christelle Langevin, François Treussart, Treussart, François, APPEL À PROJETS TRANSNATIONAL SUR LA NANOMÉDECINE - - MoDiaNo2018 - ANR-18-ENM3-0002 - ERAnet EURONANOMED - VALID, Enabling Vision Restoration - - FOReSIGHT2018 - ANR-18-IAHU-0001 - IAHU - VALID, Comprendre comment l'homéostasie du transport axonal est médiée par le 'tubulin code' - - CodeAx2020 - ANR-20-CE13-0011 - AAPG2020 - VALID, iReelAx : Analyse interspécifique des fonctions de Reelin dans le câblage des circuits et les troubles neurodéveloppementaux - - iReelAx2019 - ANR-19-CE16-0017 - AAPG2019 - VALID, Idex Paris-Saclay - - IPS2011 - ANR-11-IDEX-0003 - IDEX - VALID, Laboratoire Lumière, Matière et Interfaces (LuMIn), CentraleSupélec-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Plateforme d'Infectiologie Expérimentale des Rongeurs et Poissons (IERP (UE 0907)), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Virologie et Immunologie Moléculaires (VIM (UR 0892)), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de la Vision, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), VIB Center for Brain and Disease Research, Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), Laboratoire de physique de la matière condensée (LPMC), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS), MITI CNRS- Fondation pour la recherche médicale (grant #MND202003011485)- FWO Fellowship (#12Y9120N), ANR-18-ENM3-0002,MoDiaNo(2018), ANR-18-IAHU-0001,FOReSIGHT,Enabling Vision Restoration(2018), ANR-20-CE13-0011,CodeAx,Comprendre comment l'homéostasie du transport axonal est médiée par le 'tubulin code'(2020), ANR-19-CE16-0017,iReelAx,iReelAx : Analyse interspécifique des fonctions de Reelin dans le câblage des circuits et les troubles neurodéveloppementaux(2019), and ANR-11-IDEX-0003,IPS,Idex Paris-Saclay(2011)
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[PHYS.PHYS.PHYS-OPTICS] Physics [physics]/Physics [physics]/Optics [physics.optics] ,[PHYS.PHYS.PHYS-OPTICS]Physics [physics]/Physics [physics]/Optics [physics.optics] ,second harmonic generation ,[SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging ,General Engineering ,General Physics and Astronomy ,nonlinear nanocrystal ,disease models ,molecular motor ,[SDV.IB.IMA] Life Sciences [q-bio]/Bioengineering/Imaging ,zebrafish larva ,[SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC] ,General Materials Science ,two-photon microscopy ,[SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC] ,axonal transport ,single nanoparticle tracking - Abstract
International audience; Cargo transport by molecular motors along microtubules is essential for the function of eucaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and of the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈ 90 μ m×60 μ m size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole, and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having non-linear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate, and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated to disease models.
- Published
- 2022
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