Quantification du transport intraneuronal par suivi de nanodiamants fluorescents. Application à l’étude de l’impact fonctionnel de facteurs de risque génétiques associés aux maladies neuropsychiatriques.

The identification of molecular biomarkers of brain diseases as diverse as autism, schizophrenia and Alzheimer’s disease, is of crucial importance not only for an objective diagnosis but also to monitor response to treatments. The establishment and maintenance of sub-cellular neuronal functions, such as synaptic plasticity, are highly dependent on intracellular transport, which is essential to deliver important materials to specific locations. Abnormalities in such active transport are thought to be partly responsible for synaptic plasticity and neuronal morphology impairment found in many neuropsychiatric and neurodegenerative diseases. This thesis reports (i) the development of a quantification technic of intraneuronal transport based on fluorescent nanodiamonds (fNDs) tracking; (ii) the application of this simple and minimally invasive approach to the functional analysis of neuropsychiatric disease-related genetic variants.This manuscript falls into four chapters. The first one details the complex polygenic architecture of mental disorders and demonstrates the disease relevance of monitoring the intraneuronal transport. The second and the third chapters are dedicated to the nanodiamond-tracking assay and describe the fNDs internalisation strategies, the spatiotemporal quantitative readouts and the validation of the technic. The high brightness, the perfect photostability and the absence of cytotoxicity make fNDs a tool of choice to perform high throughput long-term bioimaging at high spatiotemporal resolution. Finally, in the fourth chapter, we apply this new functional analysis method to study the effect of genetic variants associated to autism and schizophrenia. We established transgenic mouse lines in which MARK1 and SLC25A12 genes were slightly overexpressed, and AAV-shRNA to induce AUTS2 gene haploinsufficiency. Our molecular diagnosis assay proves sufficiently sensitive to detect fine changes in intraneuronal transport dynamic, paving the way for future development in translational nanomedicine.L’identification de biomarqueurs des maladies mentales telles que l’autisme, la schizophrénie ou la maladie d’Alzheimer, est d’une importance capitale non seulement pour établir un diagnostic objectif, mais aussi pour suivre l’effet des traitements. La création et le maintien de fonctions neuronales sub-cellulaires, telle que la plasticité synaptique, sont fortement dépendants du transport intraneuronal, essentiel pour acheminer d’importants composants à des positions spécifiques. Un transport actif défaillant semble être partiellement responsable d’anomalies de la plasticité synaptique et de la morphologie neuronale présentes dans de nombreuses maladies neuropsychiatriques. Cette thèse décrit (i) la mise au point d’une méthode de quantification du transport intraneuronal reposant sur le suivi de nanoparticules de diamants fluorescents (fNDs); (ii) l’application de cette technique simple et faiblement invasive à l’analyse fonctionnelle de variants génétiques associés à des maladies neuropsychiatriques. Ce manuscrit comporte quatre chapitres. Le premier détaille l’architecture polygénique complexe des maladies mentales et démontre la pertinence d’étudier le transport intraneuronal. Les deuxième et troisième chapitres sont dédiés à la méthode et détaillent les stratégies d’internalisation des fNDs, les outils de quantification du transport intraneuronal et la validation de la technique. La forte brillance, la photo-stabilité parfaite et l’absence de toxicité cellulaire font des fNDs un outil de choix pour étudier la dynamique du transport intraneuronal sur une durée d’observation de plusieurs heures avec une haute résolution spatiotemporelle et une bonne puissance statistique. Enfin, dans le quatrième chapitre, nous appliquons cette nouvelle méthode d’analyse fonctionnelle pour étudier l’effet de variants génétiques associés à l’autisme et à la schizophrénie. Pour cela, nous utilisons des lignées de souris transgéniques ayant une faible surexpression des gènes MARK1 et SLC25A12, ainsi que des AAV-shRNA pour induire une haplo-insuffisance du gène AUTS2. Notre méthode de diagnostic moléculaire s’avère suffisamment sensible pour déceler des variations fines de la dynamique du transport intraneuronal, ouvrant la voie à de futurs développements en nanomédecine translationnelle

Quantification of intraneuronal transport by fluorescent nanodiamond tracking. Application to the screening of the functional impact of neuropsychiatric disease-related genetic risk factors.

L’identification de biomarqueurs des maladies mentales telles que l’autisme, la schizophrénie ou la maladie d’Alzheimer, est d’une importance capitale non seulement pour établir un diagnostic objectif, mais aussi pour suivre l’effet des traitements. La création et le maintien de fonctions neuronales sub-cellulaires, telle que la plasticité synaptique, sont fortement dépendants du transport intraneuronal, essentiel pour acheminer d’importants composants à des positions spécifiques. Un transport actif défaillant semble être partiellement responsable d’anomalies de la plasticité synaptique et de la morphologie neuronale présentes dans de nombreuses maladies neuropsychiatriques. Cette thèse décrit (i) la mise au point d’une méthode de quantification du transport intraneuronal reposant sur le suivi de nanoparticules de diamants fluorescents (fNDs); (ii) l’application de cette technique simple et faiblement invasive à l’analyse fonctionnelle de variants génétiques associés à des maladies neuropsychiatriques. Ce manuscrit comporte quatre chapitres. Le premier détaille l’architecture polygénique complexe des maladies mentales et démontre la pertinence d’étudier le transport intraneuronal. Les deuxième et troisième chapitres sont dédiés à la méthode et détaillent les stratégies d’internalisation des fNDs, les outils de quantification du transport intraneuronal et la validation de la technique. La forte brillance, la photo-stabilité parfaite et l’absence de toxicité cellulaire font des fNDs un outil de choix pour étudier la dynamique du transport intraneuronal sur une durée d’observation de plusieurs heures avec une haute résolution spatiotemporelle et une bonne puissance statistique. Enfin, dans le quatrième chapitre, nous appliquons cette nouvelle méthode d’analyse fonctionnelle pour étudier l’effet de variants génétiques associés à l’autisme et à la schizophrénie. Pour cela, nous utilisons des lignées de souris transgéniques ayant une faible surexpression des gènes MARK1 et SLC25A12, ainsi que des AAV-shRNA pour induire une haplo-insuffisance du gène AUTS2. Notre méthode de diagnostic moléculaire s’avère suffisamment sensible pour déceler des variations fines de la dynamique du transport intraneuronal, ouvrant la voie à de futurs développements en nanomédecine translationnelle.The identification of molecular biomarkers of brain diseases as diverse as autism, schizophrenia and Alzheimer’s disease, is of crucial importance not only for an objective diagnosis but also to monitor response to treatments. The establishment and maintenance of sub-cellular neuronal functions, such as synaptic plasticity, are highly dependent on intracellular transport, which is essential to deliver important materials to specific locations. Abnormalities in such active transport are thought to be partly responsible for synaptic plasticity and neuronal morphology impairment found in many neuropsychiatric and neurodegenerative diseases. This thesis reports (i) the development of a quantification technic of intraneuronal transport based on fluorescent nanodiamonds (fNDs) tracking; (ii) the application of this simple and minimally invasive approach to the functional analysis of neuropsychiatric disease-related genetic variants.This manuscript falls into four chapters. The first one details the complex polygenic architecture of mental disorders and demonstrates the disease relevance of monitoring the intraneuronal transport. The second and the third chapters are dedicated to the nanodiamond-tracking assay and describe the fNDs internalisation strategies, the spatiotemporal quantitative readouts and the validation of the technic. The high brightness, the perfect photostability and the absence of cytotoxicity make fNDs a tool of choice to perform high throughput long-term bioimaging at high spatiotemporal resolution. Finally, in the fourth chapter, we apply this new functional analysis method to study the effect of genetic variants associated to autism and schizophrenia. We established transgenic mouse lines in which MARK1 and SLC25A12 genes were slightly overexpressed, and AAV-shRNA to induce AUTS2 gene haploinsufficiency. Our molecular diagnosis assay proves sufficiently sensitive to detect fine changes in intraneuronal transport dynamic, paving the way for future development in translational nanomedicine

DYRK1A up-regulation specifically impairs a presynaptic form of long-term potentiation

article scientifique soumisChromosome 21 DYRK1A kinase has long been associated with a variety of psychiatric diseases including Down Syndrome. We previously showed that Dyrk1A interacts with SWI/SNF (SWItch/Sucrose Non-Fermentable) nucleosome remodeling complex inducing expression changes of genes encoding key neuronal proteins. However, the functional impact of this kinase at the synapse level remains unclear. We studied a mouse model that incorporated the YAC 152F7 (570 kb) encoding six chromosome 21 genes including DYRK1A. We found that DYRK1A Interacts with the key chromatin remodelers EP300 and CREBBP. Moreover, we observed changes in the transcriptional levels of genes encoding presynaptic proteins involved in glutamate vesicle exocytosis, namely Rims1, Munc13-1, Syn2, Rab3A. This result prompted us to investigate the two main forms of long-term potentiation (LTP) required for learning and memory: the (N-methyl d-aspartate) receptor-dependent postsynaptic form versus the glutamate release-dependent presynaptic form. Interestingly, extracellular electrophysiological recordings in hippocampal slices of the YAC mouse line revealed that only the presynaptic forms of plasticity were impacted, leaving the post-synaptic form of plasticity intact. T o refine our findings, we used a mouse BAC 189N3 (152 kb) line that only triplicate the gene Dyrk1A. Again, we found that this presynaptic form of LTP is also impaired in this mouse line. This result demonstrates that abnormal up-regulation of Dyrk1A alone is sufficient to inhibit specifically the presynaptic forms of LTP. Altogether, our results suggest that impairment of DYRK1A gene dosage may impact memory precision, and therefore reinforce our mechanistic understanding of the cognitive impairment detected in this mouse model

Single particle tracking of fluorescent nanodiamonds in cells and organisms

International audienceSingle particle tracking enables nanoscale investigation of cellular processes. Fluorescent nanodiamond contains nitrogen-vacancy centers as photostable emitters. Background-free imaging and tracking are achievable with nitrogen-vacancy centers. Fluorescent nanodiamonds are suitable for long-term tracking in whole organisms

Cognitive radio for high speed railway through dynamic and opportunistic spectrum reuse

TRA2014 Transport Research Arena 2014 - Transport Solutions: from Research to Deployment - Innovate Mobility, Mobilise Innovation! 14-17 Apr 2014 Paris La Défense (France)International audienceThe integration of all the heterogeneous wireless networks deployed along European railway lines constitutes a key technical challenge to improve global efficiency of railway system. Emerging Cognitive Radio (CR) technologies can answer this challenge. The paper presents the first results obtained in the ANR project CORRIDOR (Cognitive Radio for High Speed Railway through Dynamic and Opportunistic spectrum Reuse) which is the first research project devoted to CR systems for Railway in France, taking into account very demanding railway applications: GSM-R which is the radio link of the ERTMS system, and other operational needs such as video surveillance of the inside of trains, data related to maintenance and Internet on board for passengers

Cognitive radio for high speed railway through dynamic and opportunistic spectrum reuse

International audienceThe integration of all the heterogeneous wireless networks deployed along European railway lines constitutes a key technical challenge to improve global efficiency of railway system. Emerging Cognitive Radio (CR) technologies can answer this challenge. The paper presents the first results obtained in the ANR project CORRIDOR (Cognitive Radio for High Speed Railway through Dynamic and Opportunistic spectrum Reuse) which is the first research project devoted to CR systems for Railway in France, taking into account very demanding railway applications: GSM-R which is the radio link of the ERTMS system, and other operational needs such as video surveillance of the inside of trains, data related to maintenance and Internet on board for passengers

Cognitive radio for high speed railway through dynamic and opportunistic spectrum reuse

International audienceThe integration of all the heterogeneous wireless networks deployed along European railway lines constitutes a key technical challenge to improve global efficiency of railway system. Emerging Cognitive Radio (CR) technologies can answer this challenge. The paper presents the first results obtained in the ANR project CORRIDOR (Cognitive Radio for High Speed Railway through Dynamic and Opportunistic spectrum Reuse) which is the first research project devoted to CR systems for Railway in France, taking into account very demanding railway applications: GSM-R which is the radio link of the ERTMS system, and other operational needs such as video surveillance of the inside of trains, data related to maintenance and Internet on board for passengers

Cognitive radio for high speed railway through dynamic and opportunistic spectrum reuse

TRA2014 Transport Research Arena 2014 - Transport Solutions: from Research to Deployment - Innovate Mobility, Mobilise Innovation! 14-17 Apr 2014 Paris La Défense (France)International audienceThe integration of all the heterogeneous wireless networks deployed along European railway lines constitutes a key technical challenge to improve global efficiency of railway system. Emerging Cognitive Radio (CR) technologies can answer this challenge. The paper presents the first results obtained in the ANR project CORRIDOR (Cognitive Radio for High Speed Railway through Dynamic and Opportunistic spectrum Reuse) which is the first research project devoted to CR systems for Railway in France, taking into account very demanding railway applications: GSM-R which is the radio link of the ERTMS system, and other operational needs such as video surveillance of the inside of trains, data related to maintenance and Internet on board for passengers