Exosomes neuronaux (rôle dans le passage intercellulaire de protéines et d'ARN)

Les exosomes sont des vésicules d'origine endocytaire sécrétées par les cellules dans leur environnement après fusion des endosomes multivésiculés avec la membrane plasmique. Ils représentent un nouveau moyen de communication cellulaire par le transfert intercellulaire de protéines, de lipides et d'ARN. Dans le laboratoire, nous nous intéressons aux rôles que pourraient jouer les exosomes neuronaux dans le système nerveux central. Nous avons montré que les neurones matures sécrètent des exosomes. Nous avons mis en évidence que cette sécrétion est directement reliée à l'activité synaptique glutamatergique et à une entrée de Ca2+. Nous avons également découvert que la partie C-terminale de la chaîne lourde de la toxine du tétanos peut être sécrétée par voie exosomale. Nous avons observé que les exosomes la contenant sont repris par des neurones en culture. Un tel cargo semble d'ailleurs influencer le devenir des exosomes. De plus, pour étudier la recapture des exosomes, nous avons utilisé des exosomes de cellules N2a exprimant la tétraspanine CD63 fusionnée à la GFP. En incubant des neurones d'hippocampe avec des exosomes GFP-CD63, nous sommes parvenus à démontrer qu'ils étaient endocytés par les neurones receveurs. Cependant, bien que les exosomes semblent avoir été internalisés, nos résultats suggèrent que leur trafic serait indépendant de la voie endocytaire classique. Enfin, nous nous sommes intéressé au contenu en ARN des exosomes de N2a et de neurones. Nous avons démontré qu'ils contenaient majoritairement des ARN courts (<= 200 nucléotides) parmi lesquels, les microARN 132 et 138. Les microARN sont de puissants régulateurs de l'expression génique. Leur transfert, via les exosomes, représenterait une nouvelle voie de régulation très fine et avec un impact conséquent sur le fonctionnement du système nerveux. Les exosomes neuronaux pourraient donc jouer un rôle dans la physiologie normale de la synapse, en permettant l'échange d'ARN et de récepteurs aux neurotransmetteurs entre neurones. Ils pourraient également être impliqués dans la propagation de protéines pathogènes comme la toxine du tétanos et la propagation de certaines maladies neurodégénératives comme Alzheimer et Creutzfeldt-Jacob. L'ensemble de nos résultats suggère que les exosomes joueraient un rôle-clé dans le système nerveux central, de par leur implication dans des processus physiologiques et pathologiques.Exosomes are vesicles of endocytic origin released by cells into their environment on fusion of multivesicular endosomes with the plasma membrane. They represent a novel mechanism of cell communication by intercellular transfer of proteins, lipids and RNAs. In our laboratory, we are interested in the roles neuronal exosomes could play in the central nervous system. We first showed that mature neurons secrete exosomes and that this is regulated by synaptic glutamatergic activity and by Ca2+ influx. We next demonstrated that the C-terminal part of the tetanus toxin heavy chain can be released in association with neuronal exosomes which can then be taken up by other neurons. Moreover, such a cargo seems to influence the actual fate of the exosome. In order to further examine exosome reuptake, we used exosomes from N2a cells expressing the tetraspanin CD63 fused to the green fluorescent protein, GFP. By incubating cultured hippocampal neurons with GFP-CD63 exosomes, we succeeded in proving that they were found inside the recipient neurons. However, although exosomes are internalized, our results suggest that their traffic is independent of the classical endosomal pathway. We also studied the RNAs contained in the N2a and neuronal exosomes. These were mainly short RNAs (<= 200 nucleotides) including microRNAs 132 and 138. MicroRNAs are key regulators of gene expression. Their transfer by exosomes could represent a new way for fine regulation with a potentially powerful impact on the nervous system. Neuronal exosomes could play a crucial role in the normal physiology of synapses, by allowing the exchange of RNAs and neurotransmitter receptors between neurons. They could also propagate pathogenic proteins such as tetanus toxin and be involved in neurodegenerative disorders such as Alzheimer's and Creutzfeldt-Jacob's diseases. Altogether, our results pave the way towards the demonstration that exosomes play an important part in the functioning of the central nervous system via their involvement in physiological and pathological processes.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Emerging Role of Neuronal Exosomes in the Central Nervous System

Exosomes are small extracellular vesicles, which stem from endosomes fusing with the plasma membrane, and can be recaptured by receiving cells. They contain lipids, proteins, and RNAs able to modify the physiology of receiving cells. Functioning of the brain relies on intercellular communication between neural cells. These communications can modulate the strength of responses at sparse groups of specific synapses, to modulate circuits underlying associations and memory. Expression of new genes must then follow to stabilize the long-term modifications of the synaptic response. Local changes of the physiology of synapses from one neuron driven by another, have so far been explained by classical signal transduction to modulate transcription, translation, and posttranslational modifications. In vitro evidence now demonstrates that exosomes are released by neurons in a way depending on synaptic activity; these exosomes can be retaken by other neurons suggesting a novel way for inter-neuronal communication. The efficacy of inter-neuronal transfer of biochemical information allowed by exosomes would be far superior to that of direct cell-to-cell contacts or secreted soluble factors. Indeed, lipids, proteins, and RNAs contained in exosomes secreted by emitting neurons could directly modify signal transduction and protein expression in receiving cells. Exosomes could thus represent an ideal mechanism for inter-neuronal transfer of information allowing anterograde and retrograde signaling across synapses necessary for plasticity. They might also allow spreading across the nervous system of pathological proteins like PrPsc, APP fragments, phosphorylated Tau, or Alpha-synuclein

Exosomes secreted by cortical neurons upon glutamatergic synapse activation specifically interact with neurons

Exosomes are nano-sized vesicles of endocytic origin released into the extracellular space upon fusion of multivesicular bodies with the plasma membrane. Exosomes represent a novel mechanism of cell–cell communication allowing direct transfer of proteins, lipids and RNAs. In the nervous system, both glial and neuronal cells secrete exosomes in a way regulated by glutamate. It has been hypothesized that exosomes can be used for interneuronal communication implying that neuronal exosomes should bind to other neurons with some kind of specificity. Here, dissociated hippocampal cells were used to compare the specificity of binding of exosomes secreted by neuroblastoma cells to that of exosomes secreted by cortical neurons. We found that exosomes from neuroblastoma cells bind indiscriminately to neurons and glial cells and could be endocytosed preferentially by glial cells. In contrast, exosomes secreted from stimulated cortical neurons bound to and were endocytosed only by neurons. Thus, our results demonstrate for the first time that exosomes released upon synaptic activation do not bind to glial cells but selectively to other neurons suggesting that they can underlie a novel aspect of interneuronal communication

Polyglutamine-Expanded Androgen Receptor Alteration of Skeletal Muscle Homeostasis and Myonuclear Aggregation Are Affected by Sex, Age and Muscle Metabolism

Polyglutamine (polyQ) expansions in the androgen receptor (AR) gene cause spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease characterized by lower motor neuron (MN) loss and skeletal muscle atrophy, with an unknown mechanism. We generated new mouse models of SBMA for constitutive and inducible expression of mutant AR and performed biochemical, histological and functional analyses of phenotype. We show that polyQ-expanded AR causes motor dysfunction, premature death, IIb-to-IIa/IIx fiber-type change, glycolytic-to-oxidative fiber-type switching, upregulation of atrogenes and autophagy genes and mitochondrial dysfunction in skeletal muscle, together with signs of muscle denervation at late stage of disease. PolyQ expansions in the AR resulted in nuclear enrichment. Within the nucleus, mutant AR formed 2% sodium dodecyl sulfate (SDS)-resistant aggregates and inclusion bodies in myofibers, but not spinal cord and brainstem, in a process exacerbated by age and sex. Finally, we found that two-week induction of expression of polyQ-expanded AR in adult mice was sufficient to cause premature death, body weight loss and muscle atrophy, but not aggregation, metabolic alterations, motor coordination and fiber-type switch, indicating that expression of the disease protein in the adulthood is sufficient to recapitulate several, but not all SBMA manifestations in mice. These results imply that chronic expression of polyQ-expanded AR, i.e. during development and prepuberty, is key to induce the full SBMA muscle pathology observed in patients. Our data support a model whereby chronic expression of polyQ-expanded AR triggers muscle atrophy through toxic (neomorphic) gain of function mechanisms distinct from normal (hypermorphic) gain of function mechanisms

neuronal exosomes : role in the intercellular transfer of proteins and RNAs

Les exosomes sont des vésicules d'origine endocytaire sécrétées par les cellules dans leur environnement après fusion des endosomes multivésiculés avec la membrane plasmique. Ils représentent un nouveau moyen de communication cellulaire par le transfert intercellulaire de protéines, de lipides et d'ARN. Dans le laboratoire, nous nous intéressons aux rôles que pourraient jouer les exosomes neuronaux dans le système nerveux central. Nous avons montré que les neurones matures sécrètent des exosomes. Nous avons mis en évidence que cette sécrétion est directement reliée à l'activité synaptique glutamatergique et à une entrée de Ca2+. Nous avons également découvert que la partie C-terminale de la chaîne lourde de la toxine du tétanos peut être sécrétée par voie exosomale. Nous avons observé que les exosomes la contenant sont repris par des neurones en culture. Un tel cargo semble d'ailleurs influencer le devenir des exosomes. De plus, pour étudier la recapture des exosomes, nous avons utilisé des exosomes de cellules N2a exprimant la tétraspanine CD63 fusionnée à la GFP. En incubant des neurones d'hippocampe avec des exosomes GFP-CD63, nous sommes parvenus à démontrer qu'ils étaient endocytés par les neurones receveurs. Cependant, bien que les exosomes semblent avoir été internalisés, nos résultats suggèrent que leur trafic serait indépendant de la voie endocytaire classique. Enfin, nous nous sommes intéressé au contenu en ARN des exosomes de N2a et de neurones. Nous avons démontré qu'ils contenaient majoritairement des ARN courts (≤ 200 nucléotides) parmi lesquels, les microARN 132 et 138. Les microARN sont de puissants régulateurs de l'expression génique. Leur transfert, via les exosomes, représenterait une nouvelle voie de régulation très fine et avec un impact conséquent sur le fonctionnement du système nerveux. Les exosomes neuronaux pourraient donc jouer un rôle dans la physiologie normale de la synapse, en permettant l'échange d'ARN et de récepteurs aux neurotransmetteurs entre neurones. Ils pourraient également être impliqués dans la propagation de protéines pathogènes comme la toxine du tétanos et la propagation de certaines maladies neurodégénératives comme Alzheimer et Creutzfeldt-Jacob. L'ensemble de nos résultats suggère que les exosomes joueraient un rôle-clé dans le système nerveux central, de par leur implication dans des processus physiologiques et pathologiques.Exosomes are vesicles of endocytic origin released by cells into their environment on fusion of multivesicular endosomes with the plasma membrane. They represent a novel mechanism of cell communication by intercellular transfer of proteins, lipids and RNAs. In our laboratory, we are interested in the roles neuronal exosomes could play in the central nervous system. We first showed that mature neurons secrete exosomes and that this is regulated by synaptic glutamatergic activity and by Ca2+ influx. We next demonstrated that the C-terminal part of the tetanus toxin heavy chain can be released in association with neuronal exosomes which can then be taken up by other neurons. Moreover, such a cargo seems to influence the actual fate of the exosome. In order to further examine exosome reuptake, we used exosomes from N2a cells expressing the tetraspanin CD63 fused to the green fluorescent protein, GFP. By incubating cultured hippocampal neurons with GFP-CD63 exosomes, we succeeded in proving that they were found inside the recipient neurons. However, although exosomes are internalized, our results suggest that their traffic is independent of the classical endosomal pathway. We also studied the RNAs contained in the N2a and neuronal exosomes. These were mainly short RNAs (≤ 200 nucleotides) including microRNAs 132 and 138. MicroRNAs are key regulators of gene expression. Their transfer by exosomes could represent a new way for fine regulation with a potentially powerful impact on the nervous system. Neuronal exosomes could play a crucial role in the normal physiology of synapses, by allowing the exchange of RNAs and neurotransmitter receptors between neurons. They could also propagate pathogenic proteins such as tetanus toxin and be involved in neurodegenerative disorders such as Alzheimer's and Creutzfeldt-Jacob's diseases. Altogether, our results pave the way towards the demonstration that exosomes play an important part in the functioning of the central nervous system via their involvement in physiological and pathological processes

Exosomes neuronaux : rôle dans le passage intercellulaire de protéines et d'ARN

Exosomes are vesicles of endocytic origin released by cells into their environment on fusion of multivesicular endosomes with the plasma membrane. They represent a novel mechanism of cell communication by intercellular transfer of proteins, lipids and RNAs. In our laboratory, we are interested in the roles neuronal exosomes could play in the central nervous system. We first showed that mature neurons secrete exosomes and that this is regulated by synaptic glutamatergic activity and by Ca2+ influx. We next demonstrated that the C-terminal part of the tetanus toxin heavy chain can be released in association with neuronal exosomes which can then be taken up by other neurons. Moreover, such a cargo seems to influence the actual fate of the exosome. In order to further examine exosome reuptake, we used exosomes from N2a cells expressing the tetraspanin CD63 fused to the green fluorescent protein, GFP. By incubating cultured hippocampal neurons with GFP-CD63 exosomes, we succeeded in proving that they were found inside the recipient neurons. However, although exosomes are internalized, our results suggest that their traffic is independent of the classical endosomal pathway. We also studied the RNAs contained in the N2a and neuronal exosomes. These were mainly short RNAs (≤ 200 nucleotides) including microRNAs 132 and 138. MicroRNAs are key regulators of gene expression. Their transfer by exosomes could represent a new way for fine regulation with a potentially powerful impact on the nervous system. Neuronal exosomes could play a crucial role in the normal physiology of synapses, by allowing the exchange of RNAs and neurotransmitter receptors between neurons. They could also propagate pathogenic proteins such as tetanus toxin and be involved in neurodegenerative disorders such as Alzheimer's and Creutzfeldt-Jacob's diseases. Altogether, our results pave the way towards the demonstration that exosomes play an important part in the functioning of the central nervous system via their involvement in physiological and pathological processes.Les exosomes sont des vésicules d'origine endocytaire sécrétées par les cellules dans leur environnement après fusion des endosomes multivésiculés avec la membrane plasmique. Ils représentent un nouveau moyen de communication cellulaire par le transfert intercellulaire de protéines, de lipides et d'ARN. Dans le laboratoire, nous nous intéressons aux rôles que pourraient jouer les exosomes neuronaux dans le système nerveux central. Nous avons montré que les neurones matures sécrètent des exosomes. Nous avons mis en évidence que cette sécrétion est directement reliée à l'activité synaptique glutamatergique et à une entrée de Ca2+. Nous avons également découvert que la partie C-terminale de la chaîne lourde de la toxine du tétanos peut être sécrétée par voie exosomale. Nous avons observé que les exosomes la contenant sont repris par des neurones en culture. Un tel cargo semble d'ailleurs influencer le devenir des exosomes. De plus, pour étudier la recapture des exosomes, nous avons utilisé des exosomes de cellules N2a exprimant la tétraspanine CD63 fusionnée à la GFP. En incubant des neurones d'hippocampe avec des exosomes GFP-CD63, nous sommes parvenus à démontrer qu'ils étaient endocytés par les neurones receveurs. Cependant, bien que les exosomes semblent avoir été internalisés, nos résultats suggèrent que leur trafic serait indépendant de la voie endocytaire classique. Enfin, nous nous sommes intéressé au contenu en ARN des exosomes de N2a et de neurones. Nous avons démontré qu'ils contenaient majoritairement des ARN courts (≤ 200 nucléotides) parmi lesquels, les microARN 132 et 138. Les microARN sont de puissants régulateurs de l'expression génique. Leur transfert, via les exosomes, représenterait une nouvelle voie de régulation très fine et avec un impact conséquent sur le fonctionnement du système nerveux. Les exosomes neuronaux pourraient donc jouer un rôle dans la physiologie normale de la synapse, en permettant l'échange d'ARN et de récepteurs aux neurotransmetteurs entre neurones. Ils pourraient également être impliqués dans la propagation de protéines pathogènes comme la toxine du tétanos et la propagation de certaines maladies neurodégénératives comme Alzheimer et Creutzfeldt-Jacob. L'ensemble de nos résultats suggère que les exosomes joueraient un rôle-clé dans le système nerveux central, de par leur implication dans des processus physiologiques et pathologiques

Exosomes as a novel way of interneuronal communication.

International audienceExosomes are small extracellular vesicles which stem from endosomes fusing with the plasma membrane; they contain lipids, proteins and RNAs that are able to modify receiving cells. Functioning of the brain relies on synapses, and certain patterns of synaptic activity can change the strength of responses at sparse groups of synapses, to modulate circuits underlying associations and memory. These local changes of the synaptic physiology in one neuron driven by another have, so far, been explained by classical signal transduction modulating transcription, translation and post-translational modifications. We have accumulated in vitro evidence that exosomes released by neurons in a way depending on synaptic activity can be recaptured by other neurons. Some lipids, proteins and RNAs contained in exosomes secreted by emitting neurons could directly modify signal transduction and protein expression in receiving cells. Exosomes may be an ideal mechanism for anterograde and retrograde information transfer across synapses underlying local changes in synaptic plasticity. Exosomes might also participate in the spreading across the nervous system of pathological proteins such as PrPSc (abnormal disease-specific conformation of prion protein), APP (amyloid precursor protein) fragments, phosphorylated tau or α-synuclein

Reassessing the Role of Potassium in Tomato Grown with Water Shortages

Potassium (K) is closely related to plant water uptake and use and affects key processes in assimilation and growth. The aim of this work was to find out to what extent K supply and enhanced compartmentation might improve water use and productivity when tomato plants suffered from periods of water stress. Yield, water traits, gas exchange, photosynthetic rate and biomass partition were determined. When plants suffered dehydration, increasing K supply was associated with reduction in stomatal conductance and increased water contents, but failed to protect photosynthetic rate. Potassium supplements increased shoot growth, fruit setting and yield under water stress. However, increasing the K supply could not counteract the great yield reduction under drought. A transgenic tomato line with enhanced K uptake into vacuoles and able to reach higher plant K contents, still showed poor yield performance under water stress and had lower K use efficiency than the control plants. With unlimited water supply (hydroponics), plants grown in low-K showed greater root hydraulic conductivity than at higher K availability and stomatal conductance was not associated with leaf K concentration. In conclusion, increasing K supply and tissue content improved some physiological features related to drought tolerance but did not overcome yield restrictions imposed by water stress.Peer reviewe

Release of exosomes from differentiated neurons and its regulation by synaptic glutamatergic activity.

International audienceExosomes are microvesicles released into the extracellular medium upon fusion to the plasma membrane of endosomal intermediates called multivesicular bodies. They represent ways for discarding proteins and metabolites and also for intercellular transfer of proteins and RNAs. In the nervous system, it has been hypothesized that exosomes might be involved in the normal physiology of the synapse and possibly allow the trans-synaptic propagation of pathogenic proteins throughout the tissue. As a first step to validate this concept, we used biochemical and morphological approaches to demonstrate that mature cortical neurons in culture do indeed secrete exosomes. Using electron microscopy, we observed exosomes being released from somato-dendritic compartments. The endosomal origin of exosomes was demonstrated by showing that the C-terminal domain of tetanus toxin specifically endocytosed by neurons and accumulating inside multivesicular bodies, is released in the extracellular medium in association with exosomes. Finally, we found that exosomal release is modulated by glutamatergic synaptic activity, suggesting that this process might be part of normal synaptic physiology. Thus, our study paves the way towards the demonstration that exosomes take part in the physiology of the normal and pathological nervous system

In vivo RyR1 reduction in muscle triggers a core-like myopathy

International audienceAbstract Mutations in the RYR1 gene, encoding the skeletal muscle calcium channel RyR1, lead to congenital myopathies, through expression of a channel with abnormal permeability and/or in reduced amount, but the direct functional whole organism consequences of exclusive reduction in RyR1 amount have never been studied. We have developed and characterized a mouse model with inducible muscle specific RYR1 deletion. Tamoxifen-induced recombination in the RYR1 gene at adult age resulted in a progressive reduction in the protein amount reaching a stable level of 50% of the initial amount, and was associated with a progressive muscle weakness and atrophy. Measurement of calcium fluxes in isolated muscle fibers demonstrated a reduction in the amplitude of RyR1-related calcium release mirroring the reduction in the protein amount. Alterations in the muscle structure were observed, with fibers atrophy, abnormal mitochondria distribution and membrane remodeling. An increase in the expression level of many proteins was observed, as well as an inhibition of the autophagy process. This model demonstrates that RyR1 reduction is sufficient to recapitulate most features of Central Core Disease, and accordingly similar alterations were observed in muscle biopsies from Dusty Core Disease patients (a subtype of Central Core Disease), pointing to common pathophysiological mechanisms related to RyR1 reduction