Thèse Fonctionnalisation de Nanocristaux Fluorescents par des Peptides Neurotropes pour Améliorer la Mesure du Transport Axonal dans les Neurones H/F

Université Paris-Saclay GS Chimie

Master 2 en biologie avec une spécialisation en neurosciences, ou Master 2 à l'interface Biologie-Physique-Chimie, avec des compétences en biochimie, chimie organique et analytique. Le candidat devra avoir un goût pour les approches interdisciplinaires permettant de répondre à des questions biologiques.

Établissement : Université Paris-Saclay GS Chimie
École doctorale : Sciences Chimiques : Molécules, Matériaux, Instrumentation et Biosystèmes
Laboratoire de recherche : LAMBE - Laboratoire Analyse, Modélisation et Matériaux pour la Biologie et l'Environnement
Direction de la thèse : William BUCHMANN ORCID 0000000154363382
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-03-31T23:59:59

Des défauts de transport moléculaire axonal sont observés dans les maladies neurodégénératives mais on ignore à quel stade de la maladie ils apparaissent, et quel rôle ils jouent dans son développement. L'équipe du labo LuMIn (ENS Saclay) a mis au point une méthode de mesure de ce transport reposant sur l'endocytose de nanodiamants fluorescents (FND) par des neurones de souris en culture 2D, et le suivi par vidéo-microscopie de fluorescence rapide du déplacement dirigé des endosomes qui les contiennent. Pour permettre l'internalisation spécifiquement neuronale dans des organoïdes cérébraux qui possèdent une diversité cellulaire proche de celles d'un cerveau humain (présence de cellules gliales et d'astrocytes en plus des neurones), le greffage d'un peptide neurotrope (RVG29, reconnaissant spécifiquement les neurones) sur des FND a été entrepris au labo LAMBE (Univ Evry), avec l'appui de caractérisations par des techniques d'analyses physicochimiques avancées (spectrométrie de masse, spectroscopie infrarouge, XPS, biosenseur à résonance plasmonique...). Des résultats récents ont montré qu'après incubation avec les neurones de souris en cultures (LuMIn) les FND-RVG29 interagissaient plus fortement avec ces derniers en comparaison des FND-COOH non fonctionnalisés. Cependant les propriétés des mouvements observés indiquent que les FND-RVG29 ne sont pas efficacement internalisés par les neurones, pour des raisons encore à élucider. L'objectif de cette thèse consistera à poursuivre les travaux initiés avec RVG29, en employant des méthodes de couplage chimique alternatives, ainsi que d'autres peptides neurotropes, et d'autres particules optiquement actives excitables dans le proche infrarouge (e.g. nanoparticules à conversion ascendante de photon), domaine spectral bien adapté à l'imagerie en profondeur dans les tissus, transparents à ces longueurs d'onde. Il s'agira aussi d'étendre progressivement les mesures en culture à des études menées dans des modèles biologiques plus complexes, à commencer par des tranches de cerveaux de souris, puis des organoïdes cérébraux humains (disponibles au LuMIn).

Axonal transport deficits are a hallmark of neurodegenerative diseases (1), notably linked to misfolded beta-amyloid plaques and hyperphosphorylated tau tangles in Alzheimer's (2). Recent advances allow intracellular transport visualization, primarily axonal, by tracking molecular motors (3). Most in vitro studies rely on fluorescence microscopy, requiring forced conjugate internalization via pinocytosis-a method unsuitable for primary neuron cultures. An alternative involves fusing fluorescent proteins (FPs) to motors via genetic engineering, but this requires cell transfection (low yield in neurons) and may disrupt endogenous processes or motor motility (4,5). Despite these drawbacks, FP tagging remains widely used for its specificity and adaptability in vivo, especially in transparent, genetically modifiable organisms like Drosophila and zebrafish (3).
However, most studies focus on sensory-motor neurons, neglecting the central nervous system, where axonal transport is equally critical. In vivo mouse observations, though more complex, achieve a maximum temporal resolution of 1 frame/second and spatial resolution of 200 nm over 200 µm fields (6,7). This temporal resolution misses transient events (<1 s), such as pauses caused by microtubule-associated protein obstacles (e.g., tau). These limitations arise from FP photobleaching, preventing higher laser excitation. To enhance sensitivity in measuring axonal transport parameters in live brains, new methodologies must combine improved spatial and-temporal resolutions with high-throughput imaging over 100 µm-sized field of view. To this aim, photostable optically active nanocrystals were used to measure the intraneuronal transport of endosomal compartments that they labeled from inside after their spontaneous internalization by endocytosis.
Following the seminal work of Cui et al (8) using QDots as fluorescent labels to measure nerve growth factor retrograde transport, LuMIn lab team used fluorescent diamond nanocrystals (FND) of size 35 nm (emitting in the 650-750 nm wavelength range) and evidenced subtle changes in endosomal transport in cultured neurons of transgenic mouse bearing a genetic risk factor of a neuropsychiatric disease (9). LuMIn team then used the FND tracking-based assay in mouse primary neuron cultures to document the impact on intraneuronal transport of protein aggregates found in neurogenerative diseases (10).
Considering the ethical concerns pertaining to animal experiments and the lack of complexity of mouse neuron culture, LuMIn team started to explore the use of human cerebral organoids (hCO) (11,12) as an alternative model to investigate neurodevelopmental and neurodegenerative diseases (13). When properly differentiated (11), hCO consists of the same cell type diversity as found in a human brain. FND are likely to be uptaken as well (if not better) by non-neuronal cell types, than by neurons. Hence the bare FND tracking-based assay is currently unable to specifically quantify intraneuronal transport in complex neuronal.
A strategy to overcome this limitation is to graft proteins (like rabies virus glycoprotein, RVG)-derived peptides to FND and other fluorescent nanocrystals (FNC) excitable with near-infrared light (14), in order to favor their uptake by neurons in hCO, and subsequently study the nanoconjugate axonal transport in normal and disease conditions (13). Rabies virus glycoprotein (RVG) stands out among the neurotropic peptides. It is a 500 amino-acid protein covering the surface of rabies virus and responsible for its tropism for neurons, due to its affinity for at least three receptors expressed at the neuronal membrane, notably nicotinic acetylcholine receptor (nAchR), but also on muscle cells. Consequently, RVG is used to facilitate the delivery of therapeutic agents to the central nervous system. Moreover, as many neurotrophic viruses, rabies virus uses axonal transport to invade the central nervous system, which makes RVG well-suited to form a nanoconjugate with FNC for axonal transport measurement. A 29 amino acid fragment of RVG (RVG29) was identified as sufficient to bind to nAchR.
The recent results of LuMIn and LAMBE teams (in the context of a previous thesis) have shown that after incubation with mouse neurons in culture (LuMIn), FND-RVG29 interacted more strongly with them than non-functionalized FND. However, the properties of the movements observed indicate that FND-RVG29 is not effectively internalized by neurons, for reasons that remain to be elucidated.
This new thesis will continue the work initiated with RVG29, using alternative chemical coupling methods, as well as other neurotropic peptides and other optically active particles excitable in the near infrared. Among the other neurotropic peptides, we will consider Tet1, which has the binding characteristics of tetanus toxin (14).
As nanocrystals alternative to FND, we will consider up-conversion nanoparticles that were recently demonstrated as photostable emitters well suited for fast videomicroscopy (15), hence for intraneuronal transport monitoring via FNC-neurotropic peptide tracing.
We expect that the new nanoconjugate will demonstrate a high specificity to neurons in complex cerebral tissue (mouse brain slice and human cerebral organoid) and allow to extend the nanoparticle-based intraneuronal transport assay to biological systems relevant for disease modeling.