No description available.

Clearance of molecularly damaged and misfolded synaptic vesicle (SV) proteins is vital for the maintenance of healthy, functional synapses. However, this process poses significant trafficking challenges for neurons, as the majority of degradative organelles and machinery are localized in the somatodendritic compartment, far from SV pools in presynaptic terminals. Our previous work showed that SV protein degradation is mediated by the endosomal sorting complex required for transport (ESCRT) pathway in an activity-dependent manner. Moreover, we found that neuronal activity increased ESCRT protein recruitment to axons and SV pools, suggesting a novel mechanism for regulating the trafficking of this critical degradative machinery, whose localization and transport in neurons has been unexplored. Here, we characterize the axonal transport of ESCRT-0 proteins Hrs and STAM1, the first components of the ESCRT pathway, which are critical for initiating SV protein degradation. We find that Hrs- and STAM1-positive transport vesicles exhibit increased anterograde and bidirectional motility in response to neuronal activity, as well as frequent contact with SV pools. ESCRT-0 vesicles typically colocalize with early endosome marker Rab5, but their transport dynamics do not mirror those of the total Rab5 vesicle pool. Moreover, other ESCRT pathway components and effectors do not show activity-dependent changes to motility, indicating that neuronal firing specifically regulates the motility of the ESCRT-0+ subset of Rab5+ structures in axons. Finally, we identify kinesin-3 motor protein KIF13A as essential for the activity-dependent transport of ESCRT-0 vesicles as well as the degradation of SV membrane proteins. Altogether, these studies demonstrate a novel activity-dependent mechanism for mobilizing the axonal transport of a newly characterized endosomal subtype carrying ESCRT machinery. This activity-induced transport is necessary for ESCRT-mediated degradation of synaptic vesicle proteins.

At the neuronal synapse, neurotransmitter-filled synaptic vesicles (SVs) fuse with the presynaptic plasma membrane during activity. Following exocytosis, SVs must be retrieved for neurotransmission to be maintained. Several modes of SV recycling have been identified. During mild neuronal activity, clathrin-mediated endocytosis has been regarded as the dominant SV retrieval mode, however the recently identified ultrafast endocytosis mode may also be important in this condition. During elevated activity, activity-dependent bulk endocytosis (ADBE) is the dominant SV retrieval pathway. In ADBE, large invaginations are formed from the plasma membrane, which then undergo scission to create bulk endosomes. In a second distinct step, SVs bud from these endosomes and specifically repopulate the reserve SV pool. However, since its first identification, only few molecules have been shown to participate in ADBE. The aim of this PhD was to identify novel molecules and elucidate the molecular mechanism of ADBE. To achieve this, two independent biochemical approaches were designed to purify and enrich bulk endosomes from primary neuronal cultures. In the first approach, bulk endosomes and SVs were labelled with a dye, FM1-43, using a strong stimulus. Cells were broken mechanically and the post nuclear supernatant, that contains all intracellular organelles, was collected. The supernatant was then subjected to subcellular fractionation using discontinuous Nycodenz gradients. This stimulated sample was always processed in parallel with a basal sample, where no neuronal stimulus was applied, in order to visualise activity dependent FM loading. After different fractionation protocols were applied, bulk endosomes were efficiently separated from SVs, as revealed by tracking fluorescence in different fractions. The fractionation results were further validated by electron microscopy, where bulk endosomes and SVs were labelled with horseradish peroxidase and purified using the established protocol. Immunoblotting against selected SV cargo proteins from stimulated bulk endosome and SV samples, indicated the specific and preferential localisation of VAMP4 on bulk endosomes, in contrast to other SV cargo. The molecular identity of bulk endosomes was also approached by submitting the bulk endosome fractions to semi-quantitative mass spectrometry. This analysis revealed many different proteins that were identified in bulk endosome samples and quantification approaches further indicated proteins that can be localised on bulk endosomes and have a potential role in ADBE. A second magnetic isolation approach was designed, to purify bulk endosomes using a completely different methodology. In this case, bulk endosomes were specifically labelled with iron nanoparticles, which are preferentially taken up by bulk endosomes since they are larger than SVs. The cells were broken as before and post nuclear supernatant was acquired. In this case, the supernatant was submitted to magnetic isolation that separated iron beads labelled structures from all other intracellular organelles. An extensive immunoblotting analysis of magnetic bulk endosomes validated that VAMP4 and syndapin I, two essential ADBE proteins, were enriched in these purified samples. These magnetic bulk endosomes were also analysed using semi-quantitative MS and revealed many proteins with a potential role in ADBE. Significant overlap between the two independent methods was observed, further validating these approaches. Combining these two methods with bioinformatics tools allowed the identification of the molecular signature of ADBE as well as novel key candidates for this process. Specific molecules were investigated for their role in ADBE and SV recycling using a variety of different real-time fluorescent imaging assays. A major focus was on rab small GTPases. High molecular weight dextran uptake was used to specifically study the role of these proteins in ADBE, as it preferentially reports uptake via larger bulk endosomes. A pH sensitive chimeric protein, synaptophysin-pHluorin, was used to investigate the role of these proteins in CME. Additional imaging assays were used to answer emerging questions regarding the function and localisation of these targets in the presynapse. Using these approaches, rab11A and rab35 were found to promote ADBE and accelerate clathrin-mediated endocytosis. This effect was specific to high intensity stimulation, while SV exocytosis was not affected. Further research on the role of both novel and established ADBE molecules will provide key future insights into the mechanism of both bulk endosome generation/scission and subsequent SV reformation. A very promising group is rab proteins and now evidence for their implication in SV recycling is presented here. Identification and characterisation of new targets will allow to investigate the role of ADBE in neurotransmission in both physiology and pathophysiology.

Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 84-93). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Synaptic vesicle (SV) recycling in the presynapse is essential for the maintenance of neurotransmission. During mild stimulation clathrin-mediated endocytosis (CME) dominates, however during intense stimulation activity-dependent bulk endocytosis (ADBE) is the dominant form of membrane retrieval. The aim of this thesis was to determine how the signalling molecule GSK3 controlled ADBE, with the hypothesis that this enzyme was required at multiple stages of this endocytosis mode. I also hoped to identify a specific cargo for ADBE. I found that during intense action potential stimulation, a localised calcium increase is necessary for the activation of Akt, which inhibited GSK3. This activation was mediated via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. Furthermore, I found that phosphatidylinositol 4-kinaseIIα (PI4KIIα), a molecule whose abundance is regulated by GSK3, had a key role in ADBE. Specifically, I found that the absence of PI4KIIα accelerated CME but inhibited ADBE and that PI4KIIα controls CME and ADBE via distinct mechanisms. The PI4KIIα study revealed potential cross-talk between CME and ADBE. To determine whether modulation of either endocytosis mode impacts on the other, the retrieval of genetically-encoded reporters of SV cargo was monitored during intense stimulation during inhibition of either CME or ADBE. The recovery of almost all SV cargo was unaffected by ADBE inhibition but was arrested by abolishing CME. In contrast, VAMP4-pHluorin retrieval was perturbed by inhibiting ADBE and not by blocking CME. Knockdown of VAMP4 also arrested ADBE, indicating that in addition to being the first identified ADBE cargo, it is also essential for this endocytosis mode to proceed.

No description available.

1. The role of calmodulin (CaM) in transmitter release was investigated using liposomes to deliver CaM and monoclonal antibodies against CaM (antiCaM) directly into the frog motor nerve terminal. 2. Miniature endplate potentials (MEPPs) were recorded in a high K+ solution, and effects on transmitter release were monitored using estimates of the quantal release parameters m (number of quanta released), n (number of functional transmitter release sites), p (mean probability of release), and vars p (spatial variance in p). 3. Administration of CaM, but not heat-inactivated CaM, encapsulated in liposomes (1000 units ml-1) produced an increase in m (25%) that was due to an increase in n. MEPP amplitude was not altered by CaM. 4. Administration of antiCaM, but not heat-inactivated antiCaM, in liposomes (50 μl ml-1) produced a progressive decrease in m (40%) that was associated with decreases in n and p. MEPP amplitude was decreased (15%) after a 25 min lag time, suggesting a separation in time between the decreases in quantal release and quantal size. 5. Bath application of the membrane-permeable CaM antagonist W7 (28 μM) produced a gradual decrease in m (25%) that was associated with a decrease in n. W7 also produced a decrease in MEPP amplitude that paralleled the decrease in m. The decreases in MEPP size and m produced by W7 were both reversed by addition of CaM. 6. Our results suggest that CaM increases transmitter release by mobilizing synaptic vesicles at the frog motor nerve terminal.

In der synaptischen Vesikelmembran adulter Neuronen bildet Synaptobrevin mit Synaptophysin den Synaptophysin-Synaptobrevin-Komplex. Der Komplex wird im Gegensatz zum SNARE-Komplex nicht in embryonalen Membranen gebildet, sondern erst während der neuronalen Entwicklung hochreguliert. Dabei erfährt Synaptophysin wahrscheinlich eine posttranslationale Modifizierung, die durch einen niedermolekularen Faktor bewirkt wird. Der Synaptophysin-Synaptobrevin-Komplex spielt eine entscheidende Rolle innerhalb der Präsynapse bei der Bereitstellung von Synaptobrevin zur Bindung seiner SNARE-Partner an der Plasmamembran. Im Zustand erhöhter exozytotischer Aktivität der Synapse beschleunigt der Synaptophysin-Synaptobrevin-Komplex die Rekrutierung von Synaptobrevin für eine erneute Bildung des SNARE-Komplexes und ermöglicht damit schnelle Exozytose-Endozytose-Zyklen bei erhöhter präsynaptischer Stimulation. Der Synaptophysin-Synaptobrevin-Komplex und der SNARE-Komplex schließen sich gegenseitig aus. Synaptische Membranen sind aus Lipiden und Proteinen aufgebaut, welche miteinander in Wechselwirkungen stehen. Innerhalb der Membranen formieren sich Subdomänen wie Lipid Rafts die durch eine besondere Lipidzusammensetzung stabilisiert werden und mit speziellen Proteinen bevorzugt assoziieren. Durch die spezielle Organisation des Membranaufbaus können die Prozesse der Endozytose und der Exozytose zum Teil reguliert werden. Synaptische Vesikel, die zu den kleinsten Zellorganellen zählen, zeigen einen besonders hohen membranären Cholesterolgehalt. Synaptophysin ist ein integrales Membranprotein synaptischer Vesikel und konnte zusätzlich als spezifisch cholesterolbindendes Protein identifiziert werden. Durch die Assoziation mit cholesterolreichen Nanodomänen der synaptischen Vesikelmembran könnten die Funktionen von Synaptophysin bei der Membranstabilisation und im Synaptophysin-Synaptobrevin-Komplex der synaptischen Vesikelmembran beeinflusst werden. In dieser Arbeit wurde gezeigt, dass nach Cholesterolverminderung der Membranen von CHOp38-Zellen und PC12-Zellen mittels Filipin und Methyl-ß-cyclodextrin Synaptophysin in dem Detergens Triton X-100 unlöslicher wird. Die Cholesterolverminderung der Membranen von Neuronen aus Rattengehirngewebe und Hippokampuskulturen mittels Methyl-ß-cyclodextrin und Lovastatin führte weiterhin zu einer verminderten Bildung des Synaptophysin-Synaptobrevin-Komplexes in der synaptischen Vesikelmembran. Somit scheint die Cholesterolassoziation von Synaptophysin und damit die Organisation der synaptischen Vesikelproteine innerhalb von Membrandomänen entscheidend an der Regulation der Proteininteraktionen im Synaptophysin-Synaptobrevin-Komplex beteiligt zu sein. Zusätzlich trägt Synaptophysin durch seine Cholesterolbindung wahrscheinlich zur Stabilisierung des hohen Krümmungsgrades der Membran der synaptischen Vesikel bei. Die Auswirkungen des verminderten Cholesterolgehaltes auf Synaptophysin und den Synaptophysin-Synaptobrevin-Komplex konnten auch bei homozygoten Mausmutanten für die Niemann-Pick Krankheit nachgewiesen werden. Der Cholesterolgehalt synaptischer Vesikel ist also für die Bildung des Synaptophysin-Synaptobrevin-Komplexes entscheidend und beeinflusst direkt die synaptische Effizienz. / Synaptobrevin interacts with synaptophysin in membranes of adult small synaptic vesicles and forms the synaptophysin/synaptobrevin complex. In contrast to the SNARE complex the synaptophysin/synaptobrevin complex only occurs in adult rat brain but is absent in embryonic brain. Changes in the binding properties of synaptophysin are probably induced by a factor of low molecular weight and correlate with posttranslational modifications of the protein. The synaptophysin/synaptobrevin complex plays an important role within the presynaptic terminal promoting synaptobrevin to bind its SNARE partners at the plasma membrane. In times of increased synaptic activity at the synapse the synaptophysin/synaptobrevin complex accelerates the recruitment of synaptobrevin to form new SNARE complexes and allows for fast exocytotic/endocytotic cycles. The synaptophysin/synaptobrevin complex and the SNARE complex are mutually exclusive. Major constituents of synaptic membranes are lipids and proteins which are subjected to continuous interactions. Within the membrane form specialized environments known as lipid rafts that are stabilized through tightly packed lipids and proteins that associate preferentially with these domains. The characteristic organisation of membrane structures is crucial for regulating the process of endocytosis and exocytosis. Synaptic vesicles are among the smallest cell organelles and are especially enriched in cholesterol. The integral membrane protein synaptophysin in addition was identified as a major specifically cholesterol-binding protein. Lateral association with cholesterol enriched subunits of the synaptic vesicle membrane may contribute to mediate the functions of synaptophysin in stabilising membrane structures and may in part regulate synaptophysin/synaptobrevin complex formation. Here we show that depletion of the cholesterol content of CHOp38 cell and PC12 cell membranes by Filipin and Methyl-ß-cyclodextrin significantly changes the solubility of synaptophysin in non-ionic detergents like Triton X-100. After cholesterol depletion of adult rat brain and primary cultures of mouse hippocampus by Methyl-ß-cyclodextrin and the HMGCoA-reductase inhibitor Lovastatin the synaptophysin/synaptobrevin complex was seen to be downregulated. Thus, the synaptophysin/synaptobrevin interaction critically depends on high cholesterol content of the synaptic vesicle membrane. Thereby, synaptophysin likely contributes to stabilise the high membrane curvature of synaptic vesicles. The effects of cholesterol depletion on functional properties of synaptophysin and the synaptophysin/synaptobrevin complex could also be shown on homozygous littermates of the mouse model of Niemann-Pick type C disease. Our investigation indicates that the cholesterol content of synaptic vesicles appears to be important for the fusion of the synaptophysin/synaptobrevin complex and directly affects synaptic efficiency.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Les synapses chimiques sont des structures spécialisées permettant une transmission d'information unidirectionnelle, d’un élément présynaptique vers un élément postsynaptique. L’organisation des terminaisons présynaptiques permet la conversion d’un potentiel d’action en signal chimique. Elles se présentent sous la forme de varicosités axonales contenant des vésicules synaptiques (VSs) qui concentrent le neurotransmetteur (NT). Une partie des VSs sont apposées (ancrées) à une région de la membrane plasmique, la zone active (ZA ; Bennett et al. 1992, Siksou et al. 2009). La ZA est située face à l’accumulation postsynaptique des récepteurs au NT. La dépolarisation d’une terminaison par un potentiel d’action active des canaux calciques dépendants du voltage. L’influx de calcium qui en résulte entraîne la fusion d’une fraction des VSs ancrées avec la membrane plasmique en moins d’une milliseconde, permettant la libération de NT (Sabatini et Regehr 1996, Lisman et al. 2007). Une endocytose compensatoire permet ensuite la reformation de VSs (Rizzoli et Betz, 2005). Des questions se posent encore quant à ce trafic régulé. Nous avons étudié la régulation de l’ancrage des VSs et développé un outil pour analyser le cycle des VSs en microscopie électronique (ME).La première partie de mon travail de thèse a porté sur la régulation du nombre de VSs ancrées à la ZA. Notre objectif était de savoir si l’ancrage était régulé spécifiquement ou en coordination avec les autres paramètres morphologiques du bouton, et de déterminer le rôle de Rab3-Interacting Molecules (RIMs), protéines centrales de la ZA, dans cette régulation. La régulation de l’ancrage a été étudiée sur un modèle de cultures organotypiques de tranches d’hippocampe dont l’activité est bloquée 3 jours par l’application de tétrodotoxine. Ces tranches ont été immobilisées par congélation sous haute pression (CHP) pour être observées en ME sans les artefacts induits par les fixations aldéhydiques. Le blocage d’activité entraîne une augmentation du nombre de VSs ancrées, de la taille de la ZA, et du nombre de récepteurs postsynaptiques au glutamate de type GluA2. Le nombre de VSs total dans le bouton et la taille du bouton ne changent pas. En immunocytochimie Nous n’avons pas observé de modification de la quantité moyenne de protéines RIM1/2 dans les terminaisons présynaptiques sous l’effet du blocage d’activité. Enfin, les enregistrements électrophysiologiques ne révèlent pas de modification de fréquence des courants excitateurs miniatures malgré l’augmentation du nombre de VSs ancrées. Ces résultats montrent une régulation spécifique de la taille de la jonction synaptique par l’activité neuronale et indiquent que le nombre de VSs ancrées n’est par régulé par la quantité de RIM.La deuxième partie de mon travail a consisté à développer un outil d’étude du cycle des VSs. En effet, la ME ne permet pas d’observer des évènements dynamiques, tandis que la résolution spatiale des microscopes optiques est insuffisante pour observer directement les VSs. Nous avons voulu associer une stimulation optogénétique de neurones avec leur immobilisation rapide par CHP, afin de pouvoir observer en ME les VSs à des temps précis après la stimulation. Nous avons travaillé sur des cultures dissociées de neurones d’hippocampe de rat. Ces neurones ont été infectés avec un Adeno-Associated Virus exprimant une protéine, la ChannelRhodopsine2, pour les rendre activables par des stimulations lumineuses. Une collaboration avec Leica Microsystems a permis de modifier l’appareil de congélation (HPM) pour (i) stimuler les neurones dans l’HPM, et (ii) synchroniser cette stimulation avec la congélation. Ce nouvel outil devrait permettre dans le futur une analyse du trafic des VSs à très haute résolution temporelle et spatiale. / Chemical synapses are highly specialized structures that convey information unidirectionnally, from a presynaptic to a postsynaptic element. Presynaptic terminals convert action potentials into chemical signals. These axonal varicosities contain synaptic vesicles (SVs) filled with neurotransmitter (NT) molecules. A fraction of the SVs are apposed (docked) to a part of the plasma membrane called the active zone (AZ; Bennett et al. 1992, Siksou et al. 2009b). The AZ is located in front of the postsynaptic accumulation of NT receptors. Depolarization of a terminal by an AP activates voltage dependent calcium channels. The resulting calcium influx induces the fusion of a fraction of the docked SVs in less than a millisecond, which release their NT content (Sabatini & Regehr 1996, Lisman et al. 2007). New SVs are then produced through a process of compensatory endocytosis (Rizzoli & Betz, 2005). Unanswered questions remain about the mechanism of this regulated traffic of SVs. We studied the regulation of SVs docking and developed a tool to analyze the cycle of SVs with electron microscopy (EM). The first part of my PhD work was focused on the regulation of the number of docked SVs at the AZ. Our objective was to determine whether SVs docking was regulated specifically or together with other morphological parameters of the bouton. We also investigated the role of Rab3-Interacting Molecules (RIMs), central proteins of the AZ, in this regulation. We worked on organotypic culture of hippocampal slices in which we blocked neuronal activity with tetrodotoxin for 3 days. Slices were immobilized using high pressure freezing (HPF) to avoid artifacts due to chemical fixation, and studied with EM. Activity blockade induced an increase in the number of docked SVs, in the size of the AZ and in the number of GluA2 postsynaptic glutamate receptors. However, the total number of SVs in the bouton and the size of the bouton did not change. With immunocytochemistry we did not detect any change in the mean amount of RIM in presynaptic terminals after chronic activity blockade. Furthermore, electrophysiology recordings showed no increase of the mean frequency of mEPSCs despite the increase in the number of docked SVs. Together these results show a specific regulation of the size of the presynaptic junction by neuronal activity, and indicate that the amount of RIMs does not regulate the number of docked SVs. The second part of my work consisted in the development of a new tool to study the cycle of SVs. Indeed, EM does not allow the visualization of dynamic phenomenon, whereas optical microscopes do not have a sufficient spatial resolution to observe SVs with the required precision. We wanted to associate optogenetic stimulations of neurons with their rapid immobilization by HPF, in order to visualize SVs at precise moments after stimulation. We worked on rats hippocampal dissociated neurons cultures. These neurons were infected with an Adeno-Associated Virus encoding a light sensitive protein channel, the ChannelRhodopsin2, in order to be able to activate them with light stimulations. We collaborated with Leica Microsystems to modify our high pressure machine (HPM), so that we can (i) stimulate the neurons within the HPM, and (ii) synchronize this stimulation with the freezing. In the future, this new tool this system should allow us to analyze the traffic SVs with high temporal and spatial resolution.