Schwann Cell Exosomes Mediate Neuron–Glia Communication and Enhance Axonal Regeneration

The functional and structural integrity of the nervous system depends on the coordinated action of neurons and glial cells. Phenomena like synaptic activity, conduction of action potentials, and neuronal growth and regeneration, to name a few, are fine tuned by glial cells. Furthermore, the active role of glial cells in the regulation of neuronal functions is underscored by several conditions in which specific mutation affecting the glia results in axonal dysfunction. We have shown that Schwann cells (SCs), the peripheral nervous system glia, supply axons with ribosomes, and since proteins underlie cellular programs or functions, this dependence of axons from glial cells provides a new and unexplored dimension to our understanding of the nervous system. Recent evidence has now established a new modality of intercellular communication through extracellular vesicles. We have already shown that SC-derived extracellular vesicles known as exosomes enhance axonal regeneration, and increase neuronal survival after pro-degenerative stimuli. Therefore, the biology nervous system will have to be reformulated to include that the phenotype of a nerve cell results from the contribution of two nuclei, with enormous significance for the understanding of the nervous system in health and disease.     

Astrocyte–Neuron Communication: Functional Consequences

Astrocyte–neuron communication has recently been proposed as a potential mechanism participating to synaptic transmission. With the development of this concept and accumulating evidences in favor of a modulation of synaptic transmission by astrocytes, has emerged the term gliotransmission. It refers to the capacity of astrocytes to release various transmitters, such as ATP, glutamate, d-serine, and GABA in the vicinity of synapses. While the cellular mechanisms involved in gliotransmission still need to be better described and, for some, identified, the aim of more and more studies is to determine the role of astrocytes from a functional point of view. This review will summarize the principal studies that have investigated a potential role of astrocytes in the various functions regulated by the brain (sleep, breathing, perception, learning and memory…). This will allow us to highlight the similarities and discrepancies in the signaling pathways involved in the different areas of the brain related to these functions.     

Oligodendrocyte–Neuron Interactions: Impact on Myelination and Brain Function

In the past, glial cells were considered to be ‘glue’ cells whose primary role was thought to be merely filling gaps in neural circuits. However, a growing number of reports have indicated the role of glial cells in higher brain function through their interaction with neurons. Myelin was originally thought to be just a sheath structure surrounding neuronal axons, but recently it has been shown that myelin exerts effects on the conduction velocity of neuronal axons even after myelin formation. Therefore, the investigation of glial cell properties and the neuron-glial interactions is important for understanding higher brain function. Moreover, since there are many neurological disorders caused by glial abnormalities, further understanding of glial cell-related diseases and the development of effective therapeutic strategies are warranted. In this review, we focused on oligodendrocyte-neuron interactions, with particular attention on (1) axonal signals underlying oligodendrocyte differentiation and myelination, (2) neuronal activity-dependent myelination and (3) the effects of myelination on higher brain function.

     

Live Tracking of Inter-organ Communication by Endogenous Exosomes In Vivo

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Direct Transfer of ��-Synuclein from Neuron to Astroglia Causes Inflammatory Responses in Synucleinopathies

Abnormal neuronal aggregation of α-synuclein is implicated in the development of many neurological disorders, including Parkinson disease and dementia with Lewy bodies. Glial cells also show extensive α-synuclein pathology and may contribute to disease progression. However, the mechanism that produces the glial α-synuclein pathology and the interaction between neurons and glia in the disease-inflicted microenvironment remain unknown. Here, we show that α-synuclein proteins released from neuronal cells are taken up by astrocytes through endocytosis and form inclusion bodies. The glial accumulation of α-synuclein through the transmission of the neuronal protein was also demonstrated in a transgenic mouse model expressing human α-synuclein. Furthermore, astrocytes that were exposed to neuronal α-synuclein underwent changes in the gene expression profile reflecting an inflammatory response. Induction of pro-inflammatory cytokines and chemokines correlated with the extent of glial accumulation of α-synuclein. Together, these results suggest that astroglial α-synuclein pathology is produced by direct transmission of neuronal α-synuclein aggregates, causing inflammatory responses. This transmission step is thus an important mediator of pathogenic glial responses and could qualify as a new therapeutic target.     

Neuron–transistor coupling: interpretation of individual extracellular recorded signals

The electrical coupling of randomly migrating neurons from rat explant brain-stem slice cultures to the gates of non-metallized field-effect transistors (FETs) has been investigated. The objective of our work is the precise interpretation of extracellular recorded signal shapes in comparison to the usual patch-clamp protocols to evaluate the possible use of the extracellular recording technique in electrophysiology. The neurons from our explant cultures exhibited strong voltage-gated potassium currents through the plasma membrane. With an improved noise level of the FET set-up, it was possible to record individual extracellular responses without any signal averaging. Cells were attached by patch-clamp pipettes in voltage-clamp mode and stimulated by voltage step pulses. The point contact model, which is the basic model used to describe electrical contact between cell and transistor, has been implemented in the electrical simulation program PSpice. Voltage and current recordings and compensation values from the patch-clamp measurement have been used as input data for the simulation circuit. Extracellular responses were identified as composed of capacitive current and active potassium current inputs into the adhesion region between the cell and transistor gate. We evaluated the extracellular signal shapes by comparing the capacitive and the slower potassium signal amplitudes. Differences in amplitudes were found, which were interpreted in previous work as enhanced conductance of the attached membrane compared to the average value of the cellular membrane. Our results suggest rather that additional effects like electrodiffusion, ion sensitivity of the sensors or more detailed electronic models for the small cleft between the cell and transistor should be included in the coupling model.     

Acceleration of α-Synuclein Aggregation by Exosomes

Exosomes are small vesicles released from cells into extracellular space. We have isolated exosomes from neuroblastoma cells and investigated their influence on the aggregation of α-synuclein, a protein associated with Parkinson disease pathology. Using cryo-transmission electron microscopy of exosomes, we found spherical unilamellar vesicles with a significant protein content, and Western blot analysis revealed that they contain, as expected, the proteins Flotillin-1 and Alix. Using thioflavin T fluorescence to monitor aggregation kinetics, we found that exosomes catalyze the process in a similar manner as a low concentration of preformed α-synuclein fibrils. The exosomes reduce the lag time indicating that they provide catalytic environments for nucleation. The catalytic effects of exosomes derived from naive cells and cells that overexpress α-synuclein do not differ. Vesicles prepared from extracted exosome lipids accelerate aggregation, suggesting that the lipids in exosomes are sufficient for the catalytic effect to arise. Using mass spectrometry, we found several phospholipid classes in the exosomes, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and the gangliosides GM2 and GM3. Within each class, several species with different acyl chains were identified. We then prepared vesicles from corresponding pure lipids or defined mixtures, most of which were found to retard α-synuclein aggregation. As a striking exception, vesicles containing ganglioside lipids GM1 or GM3 accelerate the process. Understanding how α-synuclein interacts with biological membranes to promote neurological disease might lead to the identification of novel therapeutic targets.     

p38α MAP Kinase Mediates Hypoxia-Induced Motor Neuron Cell Death: A Potential Target of Minocycline’s Neuroprotective Action

Hypoxia-ischemia (HI) may play a significant role in motor neuron death associated with the pathology of spinal cord injury and, perhaps, amyotrophic lateral sclerosis. The present study employs an in vitro model of HI to investigate the role of a stress kinase pathway, i.e., p38 MAP kinase, in cell death signaling in a motor neuron cell line, i.e., NSC34, subjected to oxygen-glucose deprivation (OGD). Although the neurons were essentially tolerant to either hypoxia (0.2% O₂) or low glucose (1 mM) alone, more than 60% of them died in response to combined low oxygen and low-glucose exposure. Minocycline, a semi-synthetic tetracycline known for its neuroprotective effects in models of neurodegeneration, afforded substantial (∼50%) protection against hypoxic cell death, assessed by lactate dehydrogenase release and flow cytometry, while suppressing OGD-induced p38 MAP kinase activation. An inhibitor of p38 kinase, SB203580, as well as siRNA-mediated down-regulation of p38 kinase elicited an almost complete blockade of OGD-induced cell death. The use of p38 isoform-specific siRNAs further revealed preferential involvement of the α over the β isoform of p38 MAP kinase in hypoxic neuronal cell death in our model.     

A Putative Catenin–Cadherin System Mediates Morphogenesis of the Caenorhabditis elegans Embryo

During morphogenesis of the Caenorhabditis elegans embryo, hypodermal (or epidermal) cells migrate to enclose the embryo in an epithelium and, subsequently, change shape coordinately to elongate the body (Priess, J.R., and D.I. Hirsh. 1986. Dev. Biol. 117:156– 173; Williams-Masson, E.M., A.N. Malik, and J. Hardin. 1997. Development [Camb.]. 124:2889–2901). We have isolated mutants defective in morphogenesis that identify three genes required for both cell migration during body enclosure and cell shape change during body elongation. Analyses of hmp-1, hmp-2, and hmr-1 mutants suggest that products of these genes anchor contractile actin filament bundles at the adherens junctions between hypodermal cells and, thereby, transmit the force of bundle contraction into cell shape change. The protein products of all three genes localize to hypodermal adherens junctions in embryos. The sequences of the predicted HMP-1, HMP-2, and HMR-1 proteins are related to the cell adhesion proteins α-catenin, β-catenin/Armadillo, and classical cadherin, respectively. This putative catenin–cadherin system is not essential for general cell adhesion in the C. elegans embryo, but rather mediates specific aspects of morphogenetic cell shape change and cytoskeletal organization.The morphology of the animal body and its tissues arise as embryonic cells change their shapes and/or positions (Mittenthal and Jacobson, 1990). Many of these changes are mediated by dynamic rearrangements of cytoskeletal components (Wessells et al., 1971). Cells can organize diverse patterns of microtubules and actin filaments, and movement of actin filaments by myosin proteins is thought to generate the force that drives many morphogenetic processes. An important step toward understanding the mechanical basis of morphogenesis is the identification and characterization of molecules that pattern the cytoskeleton and translate force into concerted cell movements. For cells to change shape coordinately or move relative to each other, forces generated within an individual cell must be transmitted to adhesive junctions at the plasma membrane and exerted on neighboring cells or the extracellular matrix (Gumbiner, 1996). The best characterized cell–cell junction is the adherens junction. This type of junction usually forms a subapical, beltlike structure that mechanically links the lateral surfaces of adjacent epithelial cells. Adherens junctions contain transmembrane proteins of the cadherin family that mediate homotypic adhesion. Cadherins are thought to connect to the actin cytoskeleton indirectly through the proteins α-catenin and β-catenin. Catenin–cadherin complexes also are associated with sites of contact between blastomeres in vertebrate and invertebrate embryos. In Drosophila, mice, and Xenopus, gene inactivation of catenins or cadherins disrupts general cell adhesion and apicobasal polarity of blastomeres and epithelial cells (Heasman et al., 1994; Larue et al., 1994; Haegel et al., 1995; Cox et al., 1996; Müller and Wieschaus, 1996; Kafron et al., 1997; Torres et al., 1997). Thus, it has been difficult to define direct requirements for these proteins in cytoskeletal organization and morphogenesis, although there is evidence for specific roles in tracheal cell migration (Tanaka-Matakatsu et al., 1996) and axon outgrowth (Iwai et al., 1997) in Drosophila.The Caenorhabditis elegans embryo provides a model system for studying how cells move and change shape to generate body and tissue morphologies. At hatching, the outermost cellular layer of the body consists of a monolayer of 85 epithelial cells called hypodermal cells that are linked together by adherens junctions (White, 1988). During embryogenesis, hypodermal cells are involved in two distinct processes that transform the initially ellipsoidal mass of embryonic cells into a long, thin worm; these processes are called body enclosure and body elongation (Sulston et al., 1983; Priess and Hirsh, 1986; Williams-Masson et al., 1997). The hypodermal cells are born on the dorsal surface of the embryo. As the hypodermal cells develop adherens junction connections, they begin to spread as a sheet across the embryo until the contralateral edges of the sheet meet at the ventral midline. In the anterior of the embryo, ventral hypodermal cells on the periphery of the spreading sheet develop filopodial extensions that may function to draw the contralateral edges of the sheet together (Williams-Masson et al., 1997). In the posterior of the embryo, the contralateral edges appear to be drawn together by a purse-string–like contraction that completes the enclosure process (Williams-Masson et al., 1997). In several respects, these processes are similar to epithelial cell movements described in a variety of systems, such as wound healing in vertebrates (Martin and Lewis, 1992) and dorsal closure in Drosophila (Young et al., 1993). At the completion of body enclosure in C. elegans, the apical surfaces of the hypodermal cells resemble rectangles that are elongated along the circumferential contour of the embryo''s body. These apical surfaces begin to change shape, constricting along the circumferential contour of the body and elongating along the anterior–posterior (longitudinal) axis. The coordinate changes in the shapes of the hypodermal cells appear to cause the body to decrease in circumference and to elongate about fourfold along its longitudinal axis (Sulston et al., 1983; Priess and Hirsh, 1986). Before body elongation, the apical cytoskeleton of each hypodermal cell reorganizes to form an array of parallel actin filament bundles oriented along the circumferential contour of the body (Priess and Hirsh, 1986; Costa et al., 1997). The parallel filament bundles bridge two opposing sides of each hypodermal cell, apparently connecting to the subapical adherens junction. Contraction of the filament bundles has been proposed as the force that elongates the embryo; the bundles become shorter and thicker during elongation, and drugs that disrupt actin filament organization prevent elongation. Apical constriction of cells has been shown in other systems to drive the invagination of epithelial sheets; because of the closed, cylindrical geometry of the hypodermal sheet in C. elegans, an analogous apical constriction might instead drive body elongation (Priess and Hirsh, 1986). Although the morphology and properties of the hypodermal cells strongly suggest that they mediate body elongation, almost all of the elongation-defective mutants described thus far have mutations in genes encoding muscle or basement membrane components. Body-wall muscles underlie the hypodermis, separated by a basement membrane (Hresko et al., 1994; diagram in Fig. ​Fig.88 a). Mutations in any of several genes that eliminate embryonic muscle contraction prevent elongation beyond a twofold increase in body length; this phenotype is called Pat¹ (paralyzed, arrested elongation at twofold; Williams and Waterston, 1994). Some of the genes of the Pat class have been shown to encode muscle-specific proteins. Because the muscles and myofilaments are oriented longitudinally, muscle contraction would be expected to oppose body elongation; thus, it is not yet understood why muscle function is required for complete elongation. The genes let-2 and emb-9 encode basement membrane collagens, and mutations in these genes produce elongation defects similar to those of Pat mutants (Guo et al., 1991; Sibley et al., 1993; Williams and Waterston, 1994). The only gene identified that is both required for proper body elongation and apparently expressed in hypodermal cells is let-502 (Wissmann et al., 1997). The predicted LET-502 protein is related to Rho-binding kinases, which can activate myosin light chain kinase, suggesting that LET-502 could have a role in hypodermal cells for the contraction of the array of actin filament bundles. Open in a separate windowFigure 8Models of morphogenetic forces and molecular organization at hypodermal cell junctions. (A) Oblique view of a schematic cross-section of an embryo after fusion of the dorsal hypodermal cells. CFBs are shown as thin lines, and adherens junctions are shown as thick lines. Bands of longitudinally oriented body wall muscles (m) are shown underlying the hypodermal cells. (B) Mechanical model of forces between the dorsal hypodermis and a lateral hypodermal cell. The connection between each CFB and the adherens junction (AJ) is represented as an open circle. In the lateral hypodermal cell, the connections are pulled downward by contraction of the CFBs within the lateral cell itself and pulled upward as CFBs in the dorsal cell contract. Note that the adherens junction at the two ends of the lateral cell (shown as two springs) are oriented such that they could dissipate some of the force exerted by contractions in the dorsal cell. (C) Two molecular models for the linkage between a filament (CF) in a CFB to a filament (AJF) in the adherens junction. HMR-1 is shown at the membrane contacts between two cells and associated with HMP-2. In the top cell, HMP-1 links a CF and an AJF directly; in the bottom cell, HMP-1 links different AJFs together while another factor (X) provides the link between the AJFs and CFs. To expand our understanding of the molecular basis for morphogenesis, we have isolated and characterized a group of mutants that display similar defects in embryo morphogenesis. In this paper, we present evidence that a C. elegans catenin–cadherin system mediates morphogenetic cell shape changes and specific aspects of cytoskeletal organization. We show that the genes hmp-1, hmp-2, and hmr-1 are required for the proper migration of hypodermal cells during body enclosure and for body elongation. We demonstrate that hmp-1, hmp-2, and hmr-1 can encode proteins related to α-catenin, β-catenin, and cadherin, respectively. We show that the protein products of these genes are localized to adherens junctions in the hypodermis. Our results indicate that these proteins anchor the parallel actin filament bundles to the adherens junctions in hypodermal cells and that this coupling translates the force of bundle contraction into cell shape change.     

The Role of Altered Cell–Cell Communication in Melanoma Progression

Under normal homeostasis, melanocyte growth and behaviour is tightly controlled by the surrounding keratinocytes. Keratinocytes regulate melanocyte behaviour through a complex system of paracrine growth factors and cell-cell adhesion molecules. Pathological changes, leading to development of malignant melanoma, upset this delicate homeostatic balance and can lead to altered expression of cell-cell adhesion and cell-cell communication molecules. In particular, there is a switch from the E-cadherin-mediated keratinocyte-melanocyte partnership to the N-cadherin-mediated melanoma-melanoma and melanoma-fibroblast interaction. Other changes include the alteration in the gap junctions formed between the melanocyte and keratinocyte. Changes in the connexin expression, in particular the loss of connexin 43, may result in a reduction or a loss of gap junctional activity, which is thought to contribute towards tumour progression. In the current review we describe the alterations in cell-cell adhesion and communication associated with melanoma development and progression, and discuss how a greater understanding of these processes may aid the future therapy of this disease.     

Peroxynitrite Mediates Nitric Oxide–Induced Blood–Brain Barrier Damage

Using the in vitro blood-brain barrier (BBB) model ECV304/C6, which consists of cocultures of human umbilical vein endothelial-like cells (ECV304) and rat glioma cells (C6), the role of peroxynitrite (OONO-) in nitric oxide (NO*)-mediated BBB disruption was evaluated. Endothelial cell cultures were exposed to NO* gas, in the presence or absence of the OONO- blocker FeTPPS. Separate exposure to NO* and OONO- resulted in endothelial cell cytotoxicity and a decline in barrier integrity. Unfortunately, FeTPPS induced significant detrimental effects on model BBB integrity at a concentration of 300 microM and above. At 250 microM (the highest concentration usable), FeTPPS displayed a trend toward prevention of NO* elicited perturbation of barrier integrity. Dichlorofluorescein diacetate is oxidized to fluorescent dichlorofluorescein by OONO- but only marginally by NO* or O2*-. We observed large and rapid increases in fluorescence in ECV304 preloaded cells following NO* exposure, which were blocked by FeTPPS. Furthermore, using an antinitrotyrosine antibody we detected the nitration of endothelial cell proteins following NO* exposure and conclude that NO*-mediated BBB dysfunction is predominantly elicited by OONO- and not NO*. Proposed mechanisms of NO*-mediated OONO- elicited barrier dysfunction and damage are discussed.     

Spectroscopic Characterization of the Excitation Energy Transfer in the Fucoxanthin–Chlorophyll Protein of Diatoms

We characterized the energy transfer pathways in the fucoxanthin–chlorophyll protein (FCP) complex of the diatom Cyclotella meneghiniana by conducting ultrafast transient absorption measurements. This light harvesting antenna has a distinct pigment composition and binds chlorophyll a (Chl-a), fucoxanthin and chlorophyll c (Chl-c) molecules in a 4:4:1 ratio. We find that upon excitation of fucoxanthin to its S₂ state, a significant amount of excitation energy is transferred rapidly to Chl-a. The ensuing dynamics illustrate the presence of a complex energy transfer network that also involves energy transfer from the unrelaxed or ‘hot’ intermediates. Chl-c to Chl-a energy transfer occurs on a timescale of a 100 fs. We observe no significant spectral evolution in the Chl-a region of the spectrum. We have applied global and target analysis to model the measured excited state dynamics and estimate the spectra of the states involved; the energy transfer network is discussed in relation to the pigment organization of the FCP complex.     

Nonverbal Communication and Human–Dog Interaction

Human–dog interaction relies to a large extent on nonverbal communication, and it is therefore plausible that human sensitivity to nonverbal signals affects interactions between human and dog. Experience with dogs is also likely to influence human–dog interactions, and it has been suggested that it influences human social skills. The present study investigated possible links between human nonverbal sensitivity, experience with dogs, and the quality of human–dog interactions. Two studies are reported. In study 1, 97 veterinary students took a psychometric test assessing human nonverbal sensitivity and answered a questionnaire on their experience with dogs. The data obtained were then used to investigate the relationship between experience with dogs and sensitivity to human nonverbal communication. The results did not indicate that experience with dogs improves human nonverbal sensitivity. In study 2, 16 students with high, and 15 students with low, levels of human nonverbal sensitivity were selected. Each of the 31 students interacted once with an unknown dog in a greeting situation, and these human–dog interactions were videoed. We found that a combined score of dog behaviors relating to insecurity was associated with the students' level of nonverbal sensitivity and experience with dogs: the dog showed more of the insecure behavior when interacting with students with a low level of nonverbal sensitivity and no experience with dogs than it did when interacting with students with a high level of nonverbal sensitivity (irrespective of experience with dogs).     

Interaction of aldolase with the troponin–tropomyosin complex of bovine muscle (Short Communication)

Ultracentrifugal studies of mixtures of aldolase and the troponin-tropomyosin complex from bovine muscle showed the existence of a labile interaction between these two myofibrillar constituents in imidazole buffers, pH6.8, I 0.02-0.10 (mol/l), and the suppression of the reaction by fructose 1,6-diphosphate. Analysis of the sedimentation-velocity patterns suggests the binding of more than 2 molecules of troponin-tropomyosin/molecule of aldolase. The results illustrate the necessity of considering additional or alternative sites to F-actin to account for the observed binding of aldolase to the thin filaments of skeletal muscle.     

Cyclic GMP–gated Channels in a Sympathetic Neuron Cell Line

The stimulation of IP₃ production by muscarinic agonists causes both intracellular Ca²⁺ release and activation of a voltage-independent cation current in differentiated N1E-115 cells, a neuroblastoma cell line derived from mouse sympathetic ganglia. Earlier work showed that the membrane current requires an increase in 3′,5′-cyclic guanosine monophosphate (cGMP) produced through the NO-synthase/guanylyl cyclase cascade and suggested that the cells may express cyclic nucleotide–gated ion channels. This was tested using patch clamp methods. The membrane permeable cGMP analogue, 8-br-cGMP, activates Na⁺ permeable channels in cell attached patches. Single channel currents were recorded in excised patches bathed in symmetrical Na⁺ solutions. cGMP-dependent single channel activity consists of prolonged bursts of rapid openings and closings that continue without desensitization. The rate of occurrence of bursts as well as the burst length increase with cGMP concentration. The unitary conductance in symmetrical 160 mM Na⁺ is 47 pS and is independent of voltage in the range −50 to +50 mV. There is no apparent effect of voltage on opening probability. The dose response curve relating cGMP concentration to channel opening probability is fit by the Hill equation assuming an apparent K _D of 10 μm and a Hill coefficient of 2. In contrast, cAMP failed to activate the channel at concentrations as high as 100 μm. Cyclic nucleotide gated (CNG) channels in N1E-115 cells share a number of properties with CNG channels in sensory receptors. Their presence in neuronal cells provides a mechanism by which activation of the NO/cGMP pathway by G-protein–coupled neurotransmitter receptors can directly modify Ca²⁺ influx and electrical excitability. In N1E-115 cells, Ca²⁺ entry by this pathway is necessary to refill the IP₃-sensitive intracellular Ca²⁺ pool during repeated stimulation and CNG channels may play a similar role in other neurons.