Добірка наукової літератури з теми "Synaptic adhesion proteins"

Alzheimer’s disease (AD) is a neurodegenerative brain disorder associated with the loss of synapses between neurons in the brain. Synaptic cell adhesion molecules are cell surface glycoproteins which are expressed at the synaptic plasma membranes of neurons. These proteins play key roles in formation and maintenance of synapses and regulation of synaptic plasticity. Genetic studies and biochemical analysis of the human brain tissue, cerebrospinal fluid, and sera from AD patients indicate that levels and function of synaptic cell adhesion molecules are affected in AD. Synaptic cell adhesion molecules interact with Aβ, a peptide accumulating in AD brains, which affects their expression and synaptic localization. Synaptic cell adhesion molecules also regulate the production of Aβvia interaction with the key enzymes involved in Aβformation. Aβ-dependent changes in synaptic adhesion affect the function and integrity of synapses suggesting that alterations in synaptic adhesion play key roles in the disruption of neuronal networks in AD.

The most prominent structural hallmark of the mammalian neocortical circuitry is the layer-based organization of specific cell types and synaptic inputs. Accordingly, cortical inhibitory interneurons (INs), which shape local network activity, exhibit subtype-specific laminar specificity of synaptic outputs. However, the underlying molecular mechanisms remain unknown. Here, we demonstrate that Immunoglobulin Superfamily member 11 (IgSF11) homophilic adhesion proteins are preferentially expressed in one of the most distinctive IN subtypes, namely, chandelier cells (ChCs) that specifically innervate axon initial segments of pyramidal neurons (PNs), and their synaptic laminar target. Loss-of-function experiments in either ChCs or postsynaptic cells revealed that IgSF11 is required for ChC synaptic development in the target layer. While overexpression of IgSF11 in ChCs enlarges ChC presynaptic boutons, expressing IgSF11 in nontarget layers induces ectopic ChC synapses. These findings provide evidence that synapse-promoting adhesion proteins, highly localized to synaptic partners, determine the layer-specific synaptic connectivity of the cortical IN subtype.

Current theories on the pathogenesis of autism spectrum disorders (ASD) maintain that the associated cognitive and behavioral symptoms are caused by aberrant synaptic transmission affecting specific brain circuits. Transgenic mouse models have implicated the involvement of cell adhesion proteins in synaptic dysfunction and ASD pathogenesis. Recently, Aoto et al. ( Cell 154: 75–88, 2013) has shown that alternatively spliced neurexin proteins affect the efficacy of AMPA receptor-mediated excitatory currents in both cultured neuronal networks and acute hippocampal slices constituting a potential ASD-related electrophysiological phenotype.

Research on the molecular and cellular basis of learning and memory has focused on the mechanisms that underlie the induction and expression of synaptic plasticity. There is increasing evidence that structural changes at the synapse are associated with synaptic plasticity and that extracellular matrix (ECM) components and cell adhesion molecules are associated with these changes. The functions of both groups of molecules can be regulated by proteolysis. In this article we review the roles of selected proteases and protease inhibitors in perisynaptic proteolysis of the ECM and synaptic adhesion proteins and the impact of proteolysis on synaptic modification and cognitive function.

Molecular mechanisms linking pre- and postsynaptic membranes at the interneuronal synapses are little known. We tested the cadherin adhesion system for its localization in synapses of mouse and chick brains. We found that two classes of cadherin-associated proteins, alpha N- and beta-catenin, are broadly distributed in adult brains, colocalizing with a synaptic marker, synaptophysin. At the ultrastructural level, these proteins were localized in synaptic junctions of various types, forming a symmetrical adhesion structure. These structures sharply bordered the transmitter release sites associated with synaptic vesicles, although their segregation was less clear in certain types of synapses. N-cadherin was also localized at a similar site of synaptic junctions but in restricted brain nuclei. In developing synapses, the catenin-bearing contacts dominated their junctional structures. These findings demonstrate that interneuronal synaptic junctions comprise two subdomains, transmitter release zone and catenin-based adherens junction. The catenins localized in these junctions are likely associated with certain cadherin molecules including N-cadherin, and the cadherin/ catenin complex may play a critical role in the formation or maintenance of synaptic junctions.

Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli–CASK–Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-α as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS–CASK–liprin-α complex recruits components of the synaptic release machinery to adhesive proteins of the active zone.

Synaptic pathology is observed during hypoxic events in the central nervous system in the form of altered dendrite structure and conductance changes. These alterations are rapidly reversible, on the return of normoxia, but are thought to initiate subsequent neuronal cell death. To characterize the effects of hypoxia on regulators of synaptic stability, we examined the temporal expression of cell adhesion molecules (CAMs) in synaptosomes after transient middle cerebral artery occlusion (MCAO) in mice. We focused on events preceding the onset of ischemic neuronal cell death (< 48 h). Synaptosome preparations were enriched in synaptically localized proteins and were free of endoplasmic reticulum and nuclear contamination. Electron microscopy showed that the synaptosome preparation was enriched in spheres (≈650 nm in diameter) containing secretory vesicles and postsynaptic densities. Forebrain mRNA levels of synaptically located CAMs was unaffected at 3 h after MCAO. This is contrasted by the observation of consistent downregulation of synaptic CAMs at 20 h after MCAO. Examination of synaptosomal CAM protein content indicated that certain adhesion molecules were decreased as early as 3 h after MCAO. For comparison, synaptosomal Agrn protein levels were unaffected by cerebral ischemia. Furthermore, a marked increase in the levels of p-Ctnnb1 in ischemic synaptosomes was observed. p-Ctnnb1 was detected in hippocampal fiber tracts and in cornu ammonis 1 neuronal nuclei. These results indicate that ischemia induces a dysregulation of a subset of synaptic proteins that are important regulators of synaptic plasticity before the onset of ischemic neuronal cell death.

Experience remodels cortical connectivity during developmental windows called critical periods. Experience-dependent regulation of synaptic strength during these periods establishes circuit functions that are stabilized as critical period plasticity wanes. These processes have been extensively studied in the developing visual cortex, where critical period opening and closure are orchestrated by the assembly, maturation, and strengthening of distinct synapse types. The synaptic specificity of these processes points towards the involvement of distinct molecular pathways. Attractive candidates are pre- and postsynaptic transmembrane proteins that form adhesive complexes across the synaptic cleft. These synapse-organizing proteins control synapse development and maintenance and modulate structural and functional properties of synapses. Recent evidence suggests that they have pivotal roles in the onset and closure of the critical period for vision. In this review, we describe roles of synapse-organizing adhesion molecules in the regulation of visual critical period plasticity and we discuss the potential they offer to restore circuit functions in amblyopia and other neurodevelopmental disorders.

The synapse, the minimal element required for interneuronal communication in the nervous sytems, is a structure with a great deal of plasticity, capable of undergoing changes that alter transmission strength, and even forming new connections. This property has great implications for a number of processes, including circuit formation and learning and memory. However, the proteins behind this synaptic plasticity are still not fully understood. To uncover and characterize the proteins that regulate the plastic nature of the synapse, I turned to the Drosophilalarval neuromuscular junction (NMJ), a powerful and accessible model system. I began by examining synaptic cell adhesion, as Cell Adhesion Molecules (CAMs) have long been implicated in synaptic outgrowth as well as learning and memory. CAMs have traditionally been thought of as molecules that mediate cell adhesion between the pre- and postsynaptic membrane. However, through the course of the studies presented here I demonstrate a CAM function that goes beyond simple cell adhesion, acting as a receptor that transduces adhesive signals to the intracellular space. In particular, I have demonstrated a role for the Drosophila CAM, Fasciclin II(FasII), in a signaling complex involving the Amyloid Precursor Protein-Like (APPL) and the Drosophila homolog of X11/MINT/Lin-10 (dX11). Further results show that deletion of either APPL or dX11 inhibits the FasII mediated outgrowth. These studies show that during NMJ expansion the transinteraction between FasII molecules in the pre- and postsynaptic membrane results in the recruitment of APPL and dX11 to the presynaptic cell surface, and the initiation of a signaling cascade that leads to bouton outgrowth. The next question addressed here was regarding the cytoskeletal changes that must occur during synapse remodeling. In particular I centered on the evolutionarily conserved cell polarity complex aPKC-Par3-Par6, which is know to regulate axon growth, the cell cytoskeleton during polarized cell division, and learning and memory. To understand the role of the cytoskeleton during NMJ expansion, I examined the organization of microtubules and actin during this process. Further, I identified atypical protein kinase C (aPKC) as a regulator of microtubule dynamics. I found that aPKC is required for regulating the degree of stabilization of synaptic microtubules. This stabilization requires the Microtubule Associated Protein-1B (MAP1B) homolog Futsch, which I demonstrated was required for aPKC to associate with and stabilize the microtubule cytoskeleton. The process of synaptic expansion not only requires modifications to the presynapse, but to the postsynapse as well. Previous work demonstrates that levels of the scaffolding proteins DrosophilaMembrane Associated Guanlyate Kinase (MAGUK) protein Discs-large (DLG), as well as the vertebrate homolog Postsynaptic Density-95 (PSD-95), which are concentrated at synapses, determine the size of postsynaptic membranes. To identify the underlying mechanisms of the regulation of postsynaptic size, we performed a yeast two hybrid screen, searching for DLG interacting proteins. We found a novel interaction between DLG, and a t-SNARE, GUK-interacting Syntaxin (Gtaxin; GTX), and went on to demonstrate that this interaction is required for proper postsynaptic membrane addition. Strong hypomorphic mutations in either dlg or gtx show a dramatic reduction in postsynaptic expansion. Overexpression of DLG produces an increase of synaptic GTX, as well as an increase in postsynaptic size, and an increased formation of GTX positive SNARE complexes. Taken together, these observations suggest that the MAGUK DLG regulates postsynaptic membrane addition by modulating the formation of a SNARE complex of the t-SNARE Gtaxin, and by targeting GTX to sites of postsynaptic membrane addition. In summary, the studies performed in this thesis probe a trans-synaptic adhesion based signaling complex required for presynaptic expansion, a specific pathway for dynamic microtubule stabilization required for pre- and postsynaptic expansion, and how a scaffolding protein regulates postsynaptic membrane expansion. These processes are all interconnected to maintain the efficacy of the synapse. The studies conducted revealed important information about how these processes are accomplished, and constitute an important step to elucidate the mechanisms by which synapse plasticity occurs at the level of single synaptic terminals.

L'impression protéique a initialement été utilisée pour reproduire et comprendre l’influence de la matrice extracellulaire sur les cellules et certains de leurs composants. Au cours de la dernière décennie, l'impression subcellulaire s’est développée, permettant d’étudier les interactions protéiques et leur rôle dans les voies de signalisation ainsi que dans la formation de synapses, immunologiques ou neuronales.La connexion synaptique est médiée par les protéines d’adhésion synaptique présentes de chaque côté de la synapse. En raison de la complexité de l’environnement synaptique mais également du manque de modèle in vitro permettant d’étudier la connexion synaptique dans un environnement biomimétique et contrôlé, les rôles exacts de ces protéines dans la synaptogénèse restent encore incertains. L’impression protéique subcellulaire est une solution potentielle pour combler ce manque. Pour cela, nous avons développé deux modèles biomimétiques basés sur l’impression protéique : un premier, utilisant des cellules hétérologues, permettant d’obtenir des informations sur la cinétique d’interaction des couples protéiques et ainsi de lier cela à leur fonction potentielle. Et un deuxième, utilisant des neurones primaires hippocampique, permettant de former des synapses artificielles pour étudier la nano-organisation de la synapse au cours du développement.Le système d’impression protéique PRIMO, commercialisé par Alvéole, qui co-finance cette thèse, est peu utilisé par les neuroscientifiques. En plus des objectifs biologiques, l'objectif industriel de cette thèse est de développer des méthodologies et des preuves de concept afin de démontrer les avantages et la faisabilité de la technologie PRIMO en neuroscience.En couplant notre premier modèle avec des techniques d’imagerie sur cellules vivantes (sptPALM et FRAP), nous avons pu différencier des cinétiques d’interaction entre différents couples de protéines d’adhésion synaptique mais également pour des interactions avec des protéines d’échafaudage. Une interaction labile pour SynCAM1, qui est connue pour son rôle dans la morphologie synaptique. Une forte et stable interaction pour Neuroligine1- Neurexine1β, due à la dimérisation de Neuroligine1, qui est indispensable pour la fonctionnalité de la synapse.Avec le second modèle, nous avons démontré, en présence de LRRTM2, la formation spécifique de synapses artificielles. Ces hémi-synapses présentent des caractéristiques morphologiques et fonctionnelles proches de synapses natives, avec la présence de vésicules et d’une activité calcique spontanée. Cependant, nous n’avons pas réussi à former de postsynapses artificielles avec Neurexine1β. Basés sur nos observations et une analyse bibliographique, nous avons formulé l’hypothèse que la postsynapse pourrait être le compartiment initiateur de la synaptogenèse.En conclusion, cette étude démontre : (1) que l’impression subcellulaire est un excellent modèle pour étudier la connectivité synaptique et l’adhésion de manière générale, aussi bien d’un point de vue fonctionnel qu’organisationnel. (2) Que les modèles d’hémi-synapses utilisant l’impression protéique sont plus spécifiques que les anciens modèles. (3) Que le système PRIMO ouvre de nombreuses perspectives en neurosciences via ses capacités d’impressions quantitatives
Micropatterning was initially employed to replicate and understand the influence of the extracellular matrix on cells and some of their components. Over the past decade, subcellular printing has emerged, enabling the study of protein interactions and their role in signaling pathways as well as in the formation of synaptic, immunological, or neuronal pathways.The synaptic connection is mediated by synaptic adhesion proteins present on each side of the synapse. Due to the complexity of the synaptic environment and the lack of in vitro models to study synaptic connection in a biomimetic and controlled environment, the exact roles of these proteins in synaptogenesis remain uncertain. Subcellular protein printing presents a potential solution to address this gap. For this purpose, we have developed two biomimetic models based on protein printing: a first one using heterologous cells, providing insights into the interaction kinetics of protein pairs and linking them to their potential function. And a second one using primary neurons, allowing the formation of artificial synapses to study synaptic nano-organization during development.The protein printing system PRIMO, commercialized by Alvéole, which is co-funding this thesis, is underutilized by neuroscientists. Besides these biological objectives, the industrial aim of this thesis is to develop methodologies and proofs of concept to demonstrate the advantages and feasibility of the PRIMO technology in neuroscience.By coupling our first model, based on heterologous cells, with live-cell imaging techniques (sptPALM and FRAP), we differentiated interaction kinetics among various synaptic adhesion protein pairs and also for interactions with scaffold proteins. A labile interaction was observed for SynCAM1, known for its role in synaptic morphology. A strong and stable interaction was evident for Neuroligin1/Neurexine1β due to Neuroligin1's dimerization, which is essential for synaptic functionality.With the second model using primary hippocampal neurons, we demonstrated, in the presence of LRRTM2, the specific formation of artificial synapses. These hemi-synapses exhibited morphological and functional characteristics close to native synapses, including the presence of vesicles and spontaneous calcium activity. However, we were unable to form artificial postsynapses with Neurexine1β. Based on our observations and bibliographic analysis, we hypothesize that the postsynapse could be the initiating compartment for synaptogenesis.In conclusion, this study demonstrates: (1) that subcellular printing is an excellent model to study synaptic connectivity and adhesion from both a functional and organizational perspective. (2) That models of hemi-synapses using micropatterning are more specific than previous models. (3) That the PRIMO system opens numerous perspectives in neuroscience through its quantitative printing capabilities

Several forms of monogenic autism spectrum disorders are associated to mutations in the genes encoding for the postsynaptic cell adhesion molecules, Neuroligins. The autism-linked substitution of the arginine at position 451 by a cysteine (R451C) in Neuroligin3 induces local misfolding of the extracellular domain, causing partial retention in the endoplasmic reticulum. The accumulation of misfolded proteins in the endoplasmic reticulum can eventually result in stress conditions and ultimately in the activation of the unfolded protein response. We have generated a PC12 Tet-On cell system with inducible expression of either wild type or R451C Neuroligin3. In this system, we show that the over-expression of R451C NLGN3 leads to the activation of the unfolded protein response, both in proliferative and neuronal-differentiating conditions. The knockin mouse strain expressing R451C Neuroligin3 is currently considered a model for studying a monogenic form of autism spectrum disorders. We have characterized the effect of the mutation on Neuroligin3 protein levels during development from embryonic stage E12 to postnatal day P60, observing in the knockin mice lower Neuroligin3 protein levels and a delay in the expression of the protein. We showed that the endogenous mutant Neuroligin3 is partially retained in the endoplasmic reticulum as observed in vitro. In the R451C Neuroligin3 migrates in two different bands, representing the incompletely glycosylated ER-retained protein and the mature glycosylated form that traffics to the cell membrane. We have investigated the activation of the unfolded protein response in vivo, in the R451C Neuroligin3 mouse model and showed the Unfolded protein response and autism spectrum disorders upregulation of the two main targets, BiP and CHOP, in total brain and cerebellum extracts from both adult and embryonic knockin mice. BiP protein levels and the phosphorylation of eIF2α were significantly increased only in the cerebellum of adult knockin mice in comparison to wild type, in agreement with the mRNA data. Unfolded protein response signaling has been reported to regulate synaptic function and plasticity. The AMPA-mediated glutamatergic currents were studied in the cerebellum, where we observed a significant increase in miniature excitatory synaptic currents in Purkinje cells of the knockin in comparison to the wild type mice. The final aim of this thesis was focused on selecting molecules from a library of chemical compounds, acting in correcting the defective trafficking of mutant Neuroligin3. The cellbased screening used HEK293 stably transfected with a truncated and fluorescent form of R451C. We have identified one compound active on improving selectively the trafficking of the R451C along the secretory pathway. The effects caused by this compound are promising for evaluating in vivo the rescue of the behavioral and functional phenotype described for the R451C Knockin mouse. This molecule, or molecules structurally correlated, could be used for designing therapeutic strategies for monogenic forms of autism characterized by the retention of misfolded Neuroligins within the endoplasmic reticulum. In conclusion, we provide a link, both in vitro and in vivo, between UPR activation and a form of monogenic ASD caused the R451C misfolding mutation in Neuroligin3. We have identified in the cerebellum of the knockin mouse model expressing R451C Neuroligin3, the specific brain region where UPR targets and modulators are regulated. Lately this region has been implicated in cognitive and emotional traits typical of the autistic Unfolded protein response and autism spectrum disorders phenotype. Since UPR mediators are involved in neuronal plasticity, the activation of UPR in cerebellum can lead to neuronal circuits alterations and consequently have a role in the autistic phenotype.

Transmission of information in the nervous system largely occurs via chemical synapses, which are sites of bidirectional communication. Chemical synapses have a complex morphologic and molecular organization. Presynaptic events include synthesis and vesicular storage of the neurotransmitter; trafficking, docking, and priming of the synaptic vesicles at the presynaptic active zones; calcium-dependent neurotransmitter release by exocytosis, and recycling of synaptic vesicles by endocytosis. Postsynaptic events are mediated by neurotransmitter-gated ion channels (ionotropic receptors) that mediate fast excitatory or inhibitory effects (classical neurotransmission) and G protein-coupled receptors that mediate neuromodulatory effects. The precise development and functional apposition of presynaptic and postsynaptic elements via scaffolding proteins and transsynaptic adhesion molecules assures fast and precise synaptic transmission and plasticity. Genetic disorders affecting presynaptic events may manifest with paroxysmal dyskinesia, congenital myasthenic syndrome, and some forms of familial Parkinson disease (PD). Presynaptic membrane proteins are also targets of toxic and autoimmune disorders.