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1.
Cell Mol Life Sci ; 75(4): 757-773, 2018 02.
Article in English | MEDLINE | ID: mdl-28956068

ABSTRACT

Amyloid beta peptide (Aß), the main component of senile plaques of Alzheimer's disease brains, is produced by sequential cleavage of amyloid precursor protein (APP) and of its C-terminal fragments (CTFs). An unanswered question is how amyloidogenic peptides spread throughout the brain during the course of the disease. Here, we show that small lipid vesicles called exosomes, secreted in the extracellular milieu by cortical neurons, carry endogenous APP and are strikingly enriched in CTF-α and the newly characterized CTF-η. Exosomes from N2a cells expressing human APP with the autosomal dominant Swedish mutation contain Aß peptides as well as CTF-α and CTF-η, while those from cells expressing the non-mutated form of APP only contain CTF-α and CTF-η. APP and CTFs are sorted into a subset of exosomes which lack the tetraspanin CD63 and specifically bind to dendrites of neurons, unlike exosomes carrying CD63 which bind to both neurons and glial cells. Thus, neuroblastoma cells secrete distinct populations of exosomes carrying different cargoes and targeting specific cell types. APP-carrying exosomes can be endocytosed by receiving cells, allowing the processing of APP acquired by exosomes to give rise to the APP intracellular domain (AICD). Thus, our results show for the first time that neuronal exosomes may indeed act as vehicles for the intercellular transport of APP and its catabolites.


Subject(s)
Amyloid beta-Protein Precursor/metabolism , Endocytosis , Exosomes/metabolism , Neurons/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/chemistry , Animals , Brain/metabolism , Brain/pathology , Cells, Cultured , Embryo, Mammalian , Endocytosis/physiology , Exosomes/pathology , Female , Humans , Neurons/pathology , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Pregnancy , Protein Transport , Rats
2.
Dev Cell ; 43(6): 716-730.e7, 2017 12 18.
Article in English | MEDLINE | ID: mdl-29257951

ABSTRACT

Autophagy and autophagy-related genes (Atg) have been attributed prominent roles in tumorigenesis, tumor growth, and metastasis. Extracellular vesicles called exosomes are also implicated in cancer metastasis. Here, we demonstrate that exosome production is strongly reduced in cells lacking Atg5 and Atg16L1, but this is independent of Atg7 and canonical autophagy. Atg5 specifically decreases acidification of late endosomes where exosomes are produced, disrupting the acidifying V1V0-ATPase by removing a regulatory component, ATP6V1E1, into exosomes. The effect of Atg5 on exosome production promotes the migration and in vivo metastasis of orthotopic breast cancer cells. These findings uncover mechanisms controlling exosome release and identify means by which autophagy-related genes can contribute to metastasis in autophagy-independent pathways.


Subject(s)
Autophagy-Related Protein 5/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Vacuolar Proton-Translocating ATPases/metabolism , Animals , Autophagy/physiology , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 7/genetics , Autophagy-Related Protein 7/metabolism , Cell Line, Tumor/metabolism , Endosomes/metabolism , Exosomes/metabolism , Female , Humans , Lysosomes/metabolism , Mice , Mice, Inbred BALB C , Neoplasm Metastasis , Vacuolar Proton-Translocating ATPases/genetics
3.
Methods Mol Biol ; 1545: 129-138, 2017.
Article in English | MEDLINE | ID: mdl-27943211

ABSTRACT

Exosomes are vesicles released by most cells into their environment upon fusion of multivesicular endosomes with the plasma membrane. Exosomes are vesicles of 60-100 nm in diameter, floating in sucrose at a density of ~1.15 g/mL and carrying a number of marker proteins such as Alix, Tsg101, and Flotillin-1. We use dissociated cortical neurons cultured for around two weeks as exosome-releasing cells. In these conditions, neurons make mature synapses and form networks that can be activated by physiological stimuli. Here, we describe methods to culture differentiated cortical neurons, induce exosome release by increasing glutamatergic synapse activity, and purify exosomes by differential centrifugations followed by density separation using sucrose gradients. These protocols allow purification of neuronal exosomes released within minutes of activation of glutamatergic synapses.


Subject(s)
Cell Fractionation , Cerebral Cortex/cytology , Exosomes/metabolism , Neurons/metabolism , Synaptic Vesicles/metabolism , Animals , Cell Fractionation/methods , Cells, Cultured , Glutamates/metabolism , Rats
4.
J Extracell Vesicles ; 3: 24722, 2014.
Article in English | MEDLINE | ID: mdl-25398455

ABSTRACT

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.

5.
Biochem Soc Trans ; 41(1): 241-4, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23356290

ABSTRACT

Exosomes 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.


Subject(s)
Cell Communication , Exosomes/physiology , Neurons/physiology , Humans , Neurodegenerative Diseases/physiopathology , Neuronal Plasticity , Protein Biosynthesis , Protein Processing, Post-Translational , Signal Transduction , Transcription, Genetic
7.
Neuron ; 73(2): 374-90, 2012 Jan 26.
Article in English | MEDLINE | ID: mdl-22284190

ABSTRACT

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is involved in many cellular processes, including cell growth and differentiation, immune functions and cancer. It is activated by various cytokines, growth factors, and protein tyrosine kinases (PTKs) and regulates the transcription of many genes. Of the four JAK isoforms and seven STAT isoforms known, JAK2 and STAT3 are highly expressed in the brain where they are present in the postsynaptic density (PSD). Here, we demonstrate a new neuronal function for the JAK/STAT pathway. Using a variety of complementary approaches, we show that the JAK/STAT pathway plays an essential role in the induction of NMDA-receptor dependent long-term depression (NMDAR-LTD) in the hippocampus. Therefore, in addition to established roles in cytokine signaling, the JAK/STAT pathway is involved in synaptic plasticity in the brain.


Subject(s)
Janus Kinases/metabolism , Long-Term Synaptic Depression/physiology , STAT Transcription Factors/metabolism , Signal Transduction/physiology , Synapses/metabolism , Animals , Enzyme Inhibitors/pharmacology , Hippocampus/drug effects , Hippocampus/metabolism , Long-Term Synaptic Depression/drug effects , Neurons/drug effects , Neurons/metabolism , Phosphorylation/drug effects , Phosphorylation/physiology , Rats , Signal Transduction/drug effects , Synapses/drug effects , Tyrphostins/pharmacology
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