In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activity-dependent bulk endocytosis (ADBE). Alix (ALG-2-interacting protein X/PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins which is required for clathrin-independent endocytosis in fibroblasts. Alix is expressed in neurons and concentrates at synapses during epileptic seizures. Here, we used cultured neurons to show that Alix is recruited to presynapses where it interacts with and concentrates endophilin-A during conditions triggering ADBE. Using Alix knockout (ko) neurons, we showed that this recruitment, which requires interaction with the calcium-binding protein ALG-2, is necessary for ADBE. We also found that presynaptic compartments of Alix ko hippocampi display subtle morphological defects compatible with flawed synaptic activity and plasticity detected electrophysiologically. Furthermore, mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus and reduced propagation of the epileptiform activity. These results thus show that impairment of ADBE due to the lack of neuronal Alix leads to abnormal synaptic recovery during physiological or pathological repeated stimulations.

In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activity dependent bulk endocytosis (ADBE). Alix (ALG-2 interacting protein X)/ PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins and thereby driving deformation and fission of endosomal and cell surface membranes. In fibroblasts, Alix is required for clathrin-independent endocytosis. Here, using electron microscopy, we show that synapses from mice lacking Alix have subtle defects in presynaptic compartments, translating into flawed synaptic plasticity. Using cultured neurons, we demonstrate that Alix is required for ADBE. We further demonstrate that in order to perform ADBE, Alix must be recruited to synapses by the calcium-binding protein ALG-2 and interact with endophilin-A. Finally, we show that mutant mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus. Furthermore, propagation of the epileptiform activity to the contralateral side of kainate injection is reduced. These results thus highlight Alix ko mice as an invaluable model to study the exact role of ADBE at synapses undergoing physiological or pathological stimulations.

Amyloid beta peptide (Aβ), 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 Aβ 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.

The endosomal sorting complex required for transport (ESCRT) is made of subcomplexes (ESCRT 0-III), crucial to membrane remodelling at endosomes, nuclear envelope and cell surface. ESCRT-III shapes membranes and in most cases cooperates with the ATPase VPS4 to mediate fission of membrane necks from the inside. The first ESCRT complexes mainly serve to catalyse the formation of ESCRT-III but can be bypassed by accessory proteins like the Alg-2 interacting protein-X (ALIX). In the nervous system, ALIX/ESCRT controls the survival of embryonic neural progenitors and later on the outgrowth and pruning of axons and dendrites, all necessary steps to establish a functional brain. In the adult brain, ESCRTs allow the endosomal turn over of synaptic vesicle proteins while stable ESCRT complexes might serve as scaffolds for the postsynaptic parts. The necessity of ESCRT for the harmonious function of the brain has its pathological counterpart, the mutations in CHMP2B of ESCRT-III giving rise to several neurodegenerative diseases.

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.

Alix (ALG-2 interacting protein X) drives deformation and fission of endosomal and cell surface membranes and thereby intervenes in diverse biological processes including cell proliferation and apoptosis. Using embryonic fibroblasts of Alix knock-out mice, we recently demonstrated that Alix is required for clathrin-independent endocytosis. Here we show that mice lacking Alix suffer from severe reduction in the volume of the brain which affects equally all regions examined. The cerebral cortex of adult animals shows normal layering but is reduced in both medio-lateral length and thickness. Alix controls brain size by regulating its expansion during two distinct developmental stages. Indeed, embryonic surface expansion of the Alix ko cortex is reduced because of the loss of neural progenitors during a transient phase of apoptosis occurring between E11.5 and E12.5. Subsequent development of the Alix ko cortex occurs normally until birth, when Alix is again required for the post-natal radial expansion of the cortex through its capacity to allow proper neurite outgrowth. The need of Alix for both survival of neural progenitor cells and neurite outgrowth is correlated with its role in clathrinindependent endocytosis in neural progenitors and at growth cones. Thus Alix-dependent, clathrin independent endocytosis is essential for controlling brain size.

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 similar to 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.

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.

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.

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 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.

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] is a ubiquitinous adaptor protein first described for its capacity to bind to the calcium-binding protein, ALG-2. Alix regulates neuronal death in ways involving interactions with ALG-2 and with proteins of the ESCRT (endosomal sorting complex required for transport). Even though all Alix interactors characterized to date are involved in endosomal trafficking, the genuine function of the protein in this process remains unclear. we have demonstrated recently that Alix and ALG-2 form in the presence of calcium, a complex with apical caspases and with the endocytosed death receptor TNFR1 (tumour necrosis factor a receptor 1), thus suggesting a molecular coupling between endosomes and the cell death machinery.

Alix/apoptosis-linked gene-2 (ALG-2)-interacting protein X is an adaptor protein involved in the regulation of the endolysosomal system through binding to endophilins and to endosomal sorting complexes required for transport (ESCRT) proteins, TSG101 and CHMP4b. It was first characterized as an interactor of ALG-2, a calcium-binding protein necessary for cell death, and several observations suggest a role for Alix in controlling cell death. We used electroporation in the chick embryo to test whether overexpressed wild-type or mutated Alix proteins influence cell death in vivo. We show that Alix overexpression is sufficient to induce cell death of neuroepithelial cells. This effect is strictly dependent on its capacity to bind to ALG-2. On the other hand, expression of Alix mutants lacking the ALG-2 or the CHMP4b binding sites prevents early programmed cell death in cervical motoneurons at day 4.5 of chick embryo development. This protection afforded by Alix mutants was abolished after deletion of the TSG101, but not of the endophilin, binding sites. Our results suggest that the interaction of the ALG-2/Alix complex with ESCRT proteins is necessary for naturally occurring death of motoneurons. Therefore, Alix represents a molecular link between the endolysosomal system and the cell death machinery.

Chronic intoxication by 3-nitropropionic acid in the Lewis rat reproduces many features reminiscent of Huntington's disease including behavioural alterations and cortico-striatal degeneration. In particular, in this model, striatal degeneration is accompanied by calpain activation as found in the human disease. The present study was undertaken to determine whether the expression of Alix (apoptosis linked gene-2 interacting protein), a widespread protein involved in neuronal death, would be modified in the striatum and cortex of 3NP-treated rats. The results clearly show that Alix immunoreactivity is increased in the neuronal cell bodies of the lateral striatum, where degeneration is massive. The medial striatum and the cortex that lack neurodegeneration remain only weakly labelled. This is further evidence suggesting an involvement of Alix in the events driving neuronal death.

Apoptosis-linked gene-2 interacting protein X (Alix) is thought to be involved in both cell death and vesicular trafficking. We examined Alix expression 2 h, 6 h and 24 h after triggering seizure-dependent neuronal death by i.p. kainic acid injection. In the hippocampus, intense, transient immunolabelling was observed in the strata lucidum, oriens and radiatum, areas of high synaptic activity. The similarity of this distribution to those of synaptophysin and endophilin suggests a presynaptic localisation. Alix labelling was increased in neuronal cell bodies in kainate-sensitive regions before or concomitant with the first signs of oedema and/or neuronal eosinophilia. The increase persisted 24 h after kainate-injection in CA3 and the piriform cortex which are areas with massive swelling and numerous pyknotic neurons. This suggests that Alix may play an early role in the mechanisms leading to cell death. Taken together, our results suggest that Alix may be a molecular link between synaptic functioning and neuronal death.

Alix is a cytoplasmic protein involved in both programmed cell death and endocytosis (Oncogene 21 (2002) 6801). These activities of Alix may be related to its demonstrated capacity to bind ALG-2 (Apoptosis Linked Gene-2), CIN85/SETA and endophilins (J. Biol. Chem. 275 (2000) 19275; Science 271 (1996) 521; Cell Death Differ. 6 (1999) 124; J. Biol. Chem. 274 (1999) 1533; J. Biol. Chem. 28 (2002) 29108). Here we report for the first time the developmental expression pattern of Alix protein during chick development. We show by immunochemistry that the protein appears first in the ventral part of the rostral neural tube (stage 16 HH) and then in more caudal parts, thereby following the rostro-caudal maturation of the neural tube. Later on, the protein is found in the distal part of axons as well as in the dermomyotome where it becomes restricted to the muscle progenitors. This first demonstration of Alix modulation during development suggests a role for the protein in early phases of motoneuron and muscle growth and differentiation.

During rat brain development, striatal proteoglycan (PG) expression shows specific spatio‐temporal modifications suggesting a possible role in the guidance of its dopaminergic afferents. The effects of individual glycosaminoglycans (GAGs) on dopaminergic (DA) neuronal adhesion and outgrowth were therefore studied. We tested the behavior of dissociated embryonic rat mesencephalic cells cultivated on substrate‐bound GAGs. Neuronal attachment was very limited and quantitative morphometry revealed variations in DA fiber outgrowth depending on the type and the concentration of GAG used. Next, we developed a cryoculture system to examine how neurons react toward GAGs expressed in situ. Rat brain slices from different developmental stages were used as substrates for embryonic mesencephalic explants. Preferential regions of adherence and outgrowth were observed: the striatum was found to be the most permissive, whereas the cortex was inhibitory. Western blotting experiments confirmed quantitative and qualitative changes in chondroitin sulfate (neurocan, phosphacan) and keratan sulfate (KS) containing PGs in these substrates and enzymatic digestion of GAGs before cryoculture revealed a substantial involvement of PGs in DA neuron adhesion and outgrowth. In particular, CSPGs seemed to mediate the permissive effect of the striatum, whereas KS confers an inhibitory effect to the cortex. PGs may thus be important for limiting midbrain projections to the striatum during development and for maintaining topography in the adult. © 2002 Wiley‐Liss, Inc.

Keratan sulphate proteoglycans are potentially important during development and are possible binding molecules for the lectin, peanut agglutinin, a marker for areas that are inhibitory for axonal growth in early embryos. The present study describes the spatiotemporal distributions of keratan sulphate epitopes and peanut agglutinin binding sites during organogenesis in the developing chick from E5 to hatching. The widespread distributions of these molecules did not often overlap but clearly delimited different carbohydrate compartments demonstrating that peanut agglutinin does not necessarily bind to keratan sulphate proteoglycans. These markers were mostly extracellular but keratan sulphate, in particular, was found within certain specific cells in cartilage, gonad, heart and pancreas, at certain ages. The presence of keratan sulphate in putative germ cells during their migrations and in the gonads may be of particular importance. Their distributions generally evoke modulation of adhesion allowing cell migrations or morphogenetic movements related to epitheliomesenchymal interactions, but may also suggest an involvement in axonal guidance in skin, cartilage, gut and possibly heart. Furthermore, in the kidney, peanut agglutinin binding sites seem to be related to the functional differentiation of the nephrons.

The expression of the chondroitin sulfate proteoglycan neurocan was examined in the developing striatum of the rat and compared with the distribution of dopaminergic terminals. Neurocan immunoreactivity shows a homogeneous pattern in the embryonic striatum. In the postnatal striatum, neurocan was first expressed within the matrix but not the patch compartments, and subsequently within both. These results suggest that chondroitin sulfate proteoglycans are involved in formation of connections between the substantia nigra and striatum.

The striatum of the mammalian basal ganglia is composed of two neurochemically distinct compartments termed patches and matrix that contribute overall to a mosaic organization. Glycosaminoglycans (GAGs), the sugar moieties of proteoglycans, provide specific spatio-temporal guidance cues during the development of several functional neural systems. However, their distribution within the nigrostriatal system has not been investigated yet. Here, the immunohistochemical distributions of unsulphated (C0S), 4-sulphated (C4S) and 6-sulphated chondroitin (C6S) and keratan sulphate (KS) were examined in the developing neostriatum of rat and compared with the distribution of dopaminergic terminals. All the chondroitin sulphate (CS) isomers are homogeneously expressed in the embryonic striatum. After birth, C0S and C6S reveal the striatal mosaic in being preferentially expressed within the matrix compartment and in boundaries around patches whereas the C4S epitope is present in both compartments, with a slight patchy distribution. KS expression is detected first in the patches during the early postnatal period and subsequently only in the matrix compartment. All these GAG expressions disappear as the brain matures except for C4S which remains high throughout adult life. Furthermore, studies within the developing medial forebrain bundle reveal that CS isomers, but not KS, are expressed in and around the dopamine axonal tract but show similar developmental patterns of distribution which do not appear to be specifically associated with the nigrostriatal pathway. These results suggest a possible implication of proteoglycans during the development of the striatum and may be useful for understanding the complex cellular and molecular interactions in degeneration and plasticity of the nigrostriatal circuit in Parkinson's disease.