Biomedical subjects

Antoine Triller

Publications and source records attributed to Antoine Triller.

At least 19 recordsLinked to original sources

PubMed2006

Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse.

The auditory inner hair cell (IHC) ribbon synapse operates with an exceptional temporal precision and maintains a high level of neurotransmitter release. However, the molecular mechanisms underlying IHC synaptic exocytosis are largely unknown. We studied otoferlin, a predicted C2-domain transmembrane protein, which is defective in a recessive form of human deafness. We show that otoferlin expression in the hair cells correlates with afferent synaptogenesis and find that otoferlin localizes to ribbon-associated synaptic vesicles. Otoferlin binds Ca(2+) and displays Ca(2+)-dependent interactions with the SNARE proteins syntaxin1 and SNAP25. Otoferlin deficient mice (Otof(-/-)) are profoundly deaf. Exocytosis in Otof(-/-) IHCs is almost completely abolished, despite normal ribbon synapse morphogenesis and Ca(2+) current. Thus, otoferlin is essential for a late step of synaptic vesicle exocytosis and may act as the major Ca(2+) sensor triggering membrane fusion at the IHC ribbon synapse.

Animals↗

PubMed2006

Cytoskeleton regulation of glycine receptor number at synapses and diffusion in the plasma membrane.

Lateral diffusion of neurotransmitter receptors in and out of synapses has been postulated as a core mechanism for rapid changes in receptor number at synapses during plastic processes. In this study, we have used single particle tracking to investigate how changes in glycine receptor (GlyR) lateral diffusion properties might account for changes in receptor number at synapses after disruption of the cytoskeleton in dissociated spinal cord neurons. We found that pharmacological disruption of F-actin and microtubules decreased the amount of GlyR and gephyrin, the backbone of the inhibitory postsynaptic scaffold, at synapses. F-actin and microtubule disruption increased GlyR exchanges between the synaptic and extrasynaptic membranes and decreased receptor dwell time at synapses. GlyR lateral diffusion was predominantly controlled by microtubules in the extrasynaptic membrane and by actin at synapses. Both diffusion coefficients and confinement at synapses were affected after F-actin disruption. Our results indicate that receptor exchanges between the synaptic and extrasynaptic compartments depend on the properties of both the postsynaptic differentiation and the extrasynaptic membrane. Consequently, GlyR number at synapses may be rapidly modulated by the cytoskeleton through the regulation of lateral diffusion in the plasma membrane and of receptor stabilization at synapses.

Actins↗

PubMed2006

Regulation of gephyrin assembly and glycine receptor synaptic stability.

Gephyrin is required for the formation of clusters of the glycine receptor (GlyR) in the neuronal postsynaptic membrane. It can make trimers and dimers through its N- and C-terminal G and E domains, respectively. Gephyrin oligomerization could thus create a submembrane lattice providing GlyR-binding sites. We investigated the relationships between the stability of cell surface GlyR and the ability of gephyrin splice variants to form oligomers. Using truncated and full-length gephyrins we found that the 13-amino acid sequence (cassette 5) prevents G domain trimerization. Moreover, E domain dimerization is inhibited by the gephyrin central L domain. All of the gephyrin variants bind GlyR beta subunit cytoplasmic loop with high affinity regardless of their cassette composition. Coexpression experiments in COS-7 cells demonstrated that GlyR bound to gephyrin harboring cassette 5 cannot be stabilized at the cell surface. This gephyrin variant was found to deplete synapses from both GlyR and gephyrin in transfected neurons. These data suggest that the relative expression level of cellular variants influence the overall oligomerization pattern of gephyrin and thus the turnover of synaptic GlyR.

Animals↗

PubMed2006

Modeling synaptic dynamics driven by receptor lateral diffusion.

The synaptic weight between a pre- and a postsynaptic neuron depends in part on the number of postsynaptic receptors. On the surface of neurons, receptors traffic by random motion in and out from a microstructure called the postsynaptic density (PSD). In the PSD, receptors can be stabilized at the membrane when they bind to scaffolding proteins. We propose a mathematical model to compute the postsynaptic counterpart of the synaptic weight based on receptor trafficking. We take into account the receptor fluxes at the PSD, which can be regulated by neuronal activity, and the interactions of receptors with the scaffolding molecules. Using a Markovian approach, we estimate the mean and the fluctuations of the number of bound receptors. When the number of receptors is large, a deterministic system is also derived. Moreover, these equations can be used, for example, to fit fluorescence-recovery-after-photobleaching experiments to determine, in living neurons, the chemical binding constants for the receptors/scaffolding molecules interaction at synapses.

Algorithms↗

PubMed2006

Developmental dissociation of presynaptic inhibitory neurotransmitter and postsynaptic receptor clustering in the hypoglossal nucleus.

At postsynaptic densities of mouse hypoglossal motoneurons, the proportion of glycine receptors co-clustered with GABAA receptors increases from neonatal to adult animals, suggesting that mixed synapses might play a greater role in adult synaptic inhibition. We visualized the presynaptic correlates of these developmental changes using immunocytochemistry. At P5, presynaptic terminals contained glycine and GlyT2 and/or GABA and GAD65, but at P15, the majority of inhibitory terminals contained glycine and GlyT2 only. The GABAergic component of evoked inhibitory postsynaptic currents in HMs decreased strongly between P5 and P15. Similarly, miniature inhibitory postsynaptic currents evolved from mainly glycinergic and mixed glycinergic/GABAergic events at P3-5 to predominantly glycinergic currents at P15. These results indicate that the decrease in the proportion of functional mixed inhibitory synapses with maturation results from a loss of the ability of presynaptic terminals to release both neurotransmitters during development while co-aggregation of GlyRs + GABAARs at postsynaptic loci remained.

Animals↗

PubMed2006

Activity-dependent movements of postsynaptic scaffolds at inhibitory synapses.

Dendritic spines show an activity-dependent cytoskeleton-based remodeling coupled with variations in receptor number and the functional properties of excitatory synapses. In this study, we analyzed the dynamics of gephyrin containing inhibitory postsynaptic scaffolds imaging a Venus::gephyrin (VeGe) chimera in dissociated spinal cord neurons. We provide evidence that the postsynaptic scaffolds at mature synapses display a submicrometric rapid lateral motion and are continuously moving on the dendritic shaft. This dynamic behavior is calcium dependent and is controlled by the cytoskeleton. Minute rearrangement within the gephyrin scaffold as well as the scaffold lateral displacements are F-actin dependent. The lateral movements are counteracted by microtubules. Moreover, the action of the potassium channel blocker 4-aminopyridine and receptor antagonists indicate that the dynamics of postsynaptic gephyrin scaffolds are controlled by synaptic activity.

Animals↗

PubMed2006

The development of hippocampal interneurons in rodents.

Interneurons are GABAergic neurons responsible for inhibitory activity in the adult hippocampus, thereby controlling the activity of principal excitatory cells through the activation of postsynaptic GABAA receptors. Subgroups of GABAergic neurons innervate specific parts of excitatory neurons. This specificity indicates that particular interneuron subgroups are able to recognize molecules segregated on the membrane of the pyramidal neuron. Once these specific connections are established, a quantitative regulation of their strength must be performed to achieve the proper balance of excitation and inhibition. We will review when and where interneurons are generated. We will then detail their migration toward and within the hippocampus, and the maturation of their morphological and neurochemical characteristics. We will finally review potential mechanisms underlying the development of GABAergic interneurons.

Animals↗

PubMed2006

Analysis of synaptic ultrastructure without fixative using high-pressure freezing and tomography.

Electron microscopy allows the analysis of synaptic ultrastructure and its modifications during learning or in pathological conditions. However, conventional electron microscopy uses aldehyde fixatives that alter the morphology of the synapse by changing osmolarity and collapsing its molecular components. We have used high-pressure freezing (HPF) to capture within a few milliseconds structural features without aldehyde fixative, and thus to provide a snapshot of living synapses. CA1 hippocampal area slices from P21 rats were frozen at -173 degrees C under high pressure to reduce crystal formation, and synapses on dendritic spines were analysed after cryosubstitution and embedding. Synaptic terminals were larger than after aldehyde fixation, and synaptic vesicles in these terminals were less densely packed. Small filaments linked the vesicles in subgroups. The postsynaptic densities (PSDs) exhibited filamentous projections extending into the spine cytoplasm. Tomographic analysis showed that these projections were connected with the spine cytoskeletal meshwork. Using immunocytochemistry, we found as expected GluR1 at the synaptic cleft and CaMKII in the PSD. Actin immunoreactivity (IR) labelled the cytoskeletal meshwork beneath the filamentous projections, but was very scarce within the PSD itself. ProSAP2/Shank3, cortactin and Ena/VASP-IRs were concentrated on the cytoplasmic face of the PSD, at the level of the PSD projections. Synaptic ultrastructure after HPF was different from that observed after aldehyde fixative. The boutons were larger, and filamentous components were preserved. Particularly, filamentous projections were observed linking the PSD to the actin cytoskeleton. Thus, synaptic ultrastructure can be analysed under more realistic conditions following HPF.

Aldehydes↗

PubMed2005

Target-dependent use of co-released inhibitory transmitters at central synapses.

Corelease of GABA and glycine by mixed neurons is a prevalent mode of inhibitory transmission in the vertebrate hindbrain. However, little is known of the functional organization of mixed inhibitory networks. Golgi cells, the main inhibitory interneurons of the cerebellar granular layer, have been shown to contain GABA and glycine. We show here that, in the vestibulocerebellum, Golgi cells contact both granule cells and unipolar brush cells, which are excitatory relay interneurons for vestibular afferences. Whereas IPSCs in granule cells are mediated by GABA(A) receptors only, Golgi cell inhibition of unipolar brush cells is dominated by glycinergic currents. We further demonstrate that a single Golgi cell can perform pure GABAergic inhibition of granule cells and pure glycinergic inhibition of unipolar brush cells. This specialization results from the differential expression of GABA(A) and glycine receptors by target cells and not from a segregation of GABA and glycine in presynaptic terminals. Thus, postsynaptic selection of coreleased fast transmitters is used in the CNS to increase the diversity of individual neuronal outputs and achieve target-specific signaling in mixed inhibitory networks.

Action Potentials↗

PubMed2005

Surface trafficking of receptors between synaptic and extrasynaptic membranes: and yet they do move!

Concentration of neurotransmitter receptors at synapses is thought to result from stable binding to subsynaptic scaffold proteins. Recent data on synaptic plasticity have shown that changes in synaptic strength derive partly from modification of postsynaptic receptor numbers. This has led to the notion of receptor trafficking into and out of synapses. The proposed underlying mechanisms have under-evaluated the role of extrasynaptic receptors. Recent technological advances have allowed imaging of receptor movements at the single-molecule level, and these experiments demonstrate that receptors switch at unexpected rates between extrasynaptic and synaptic localizations by lateral diffusion. Variation in receptor numbers at postsynaptic sites is therefore likely to depend on regulation of diffusion by modification of the structure of the membrane and/or by transient interactions with scaffolding proteins. This review is part of the TINS Synaptic Connectivity series.

Animals↗

PubMed2005

Modulation of glycine receptor subunits and gephyrin expression in the rat facial nucleus after axotomy.

In the last decade, numerous studies have investigated molecular changes in excitatory glutamatergic receptors in axotomized motoneurons, but few data are available concerning the modulation of inhibitory amino acid receptors. We report here the effect of axotomy on the expression of glycine receptors, gephyrin, vesicular inhibitory amino acid transporter (VIAAT) and synapsin I in rat facial motor neurons as demonstrated by in situ hybridization and immunohistochemistry. The facial nerve trunk was sectioned unilaterally and rats were killed 1, 3, 8, 30 or 60 days after surgery. We investigated the mechanisms underlying the changes in production of these proteins following axotomy by perfusing the facial nerve with colchicine or tetrodotoxin, and injecting cardiotoxin or botulinum toxin independently and unilaterally into the whisker pads of normal rats. Animals were killed 8 days later and processed for immunohistochemistry. The abundance of GlyR subunits and gephyrin fell sharply in the axotomized facial nucleus. This decrease began 1 day after axotomy and was lowest at 8 days, with protein levels returning to normal by day 60. Abnormal synapsin immunolabelling was also observed between days 8 and 60 after axotomy but we detected no change in VIAAT immunoreactivity. The effect of colchicine was similar to, but weaker than, that of axotomy. In contrast, tetrodotoxin, cardiotoxin and botulinum toxin had no significant effect. Thus, axotomy-induced changes probably resulted from a loss of trophic factor transported from the periphery or a positive injury signal, or both. They did not seem to depend on the disruption of activity.

Animals↗

PubMed2005

Tumor necrosis factor-alpha and neuronal development.

Tumor necrosis factor-alpha (TNFalpha) is a prototypic inflammatory cytokine up-regulated in most if not all neurodegenerative diseases. Many studies have reported variable roles in the adult or pathological brain. In contrast, the implication of TNFalpha in developmental neuronal cell death has been well documented in few studies. In sympathetic and trigeminal neurons, TNFalpha acts in an autocrine manner to induce immediate cell death on neurotrophic factor deprivation. In the spinal cord, TNFalpha is transiently produced by macrophages and commits motoneurons to become competent to die 2 days later. TNFalpha is also likely to induce immediate and delayed prodeath effects in adult and pathological tissues. Data obtained in embryonic systems will thus help to develop new therapeutic approaches to pathological neuronal death in adults.

Animals↗

PubMed2004

Impaired synaptic function in the microglial KARAP/DAP12-deficient mouse.

Several proteins are expressed in both immune and nervous systems. However, their putative nonimmune functions in the brain remain poorly understood. KARAP/DAP12 is a transmembrane polypeptide associated with cell-surface receptors in hematopoeitic cells. Its mutation in humans induces Nasu-Hakola disease, characterized by presenile dementia and demyelinization. However, alteration of white matter occurs months after the onset of neuropsychiatric symptoms, suggesting that other neuronal alterations occur in the early phases of the disease. We hypothesized that KARAP/DAP12 may impact synaptic function. In mice deficient for KARAP/DAP12 function, long-term potentiation was enhanced and was partly NMDA receptor (NMDAR) independent. This effect was accompanied by changes in synaptic glutamate receptor content, as detected by the increased rectification of AMPA receptor EPSCs and increased sensitivity of NMDAR EPSCs to ifenprodil. Biochemical analysis of synaptic proteins confirmed these electrophysiological data. In mutants, the AMPA receptor GluR2 subunit expression was decreased only in the postsynaptic densities but not in the whole membrane fraction, demonstrating specific impairment of synaptic receptor accumulation. Alteration of the BNDF-tyrosine kinase receptor B (TrkB) signaling in the mutant was demonstrated by the dramatic decrease of synaptic TrkB with no change in other regulatory or scaffolding proteins. Finally, KARAP/DAP12 was detected only in microglia but not in neurons, astrocytes, or oligodendrocytes. KARAP/DAP12 may thus alter microglial physiology and subsequently synaptic function and plasticity through a novel microglia-neuron interaction.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Docosahexaenoic acid protects from dendritic pathology in an Alzheimer's disease mouse model.

Learning and memory depend on dendritic spine actin assembly and docosahexaenoic acid (DHA), an essential n-3 (omega-3) polyunsaturated fatty acid (PFA). High DHA consumption is associated with reduced Alzheimer's disease (AD) risk, yet mechanisms and therapeutic potential remain elusive. Here, we report that reduction of dietary n-3 PFA in an AD mouse model resulted in 80%-90% losses of the p85alpha subunit of phosphatidylinositol 3-kinase and the postsynaptic actin-regulating protein drebrin, as in AD brain. The loss of postsynaptic proteins was associated with increased oxidation, without concomitant neuron or presynaptic protein loss. n-3 PFA depletion increased caspase-cleaved actin, which was localized in dendrites ultrastructurally. Treatment of n-3 PFA-restricted mice with DHA protected against these effects and behavioral deficits and increased antiapoptotic BAD phosphorylation. Since n-3 PFAs are essential for p85-mediated CNS insulin signaling and selective protection of postsynaptic proteins, these findings have implications for neurodegenerative diseases where synaptic loss is critical, especially AD.

Actins↗

PubMed2004

Differentiation-dependent sensitivity to apoptogenic factors in PC12 cells.

We have investigated the role of the mitochondrial pathway during cell death following serum and nerve growth factor (NGF)/dibutyryl cyclic AMP (Bt(2)cAMP) withdrawal in undifferentiated or NGF/Bt(2)cAMP-differentiated PC12 cells, respectively. Holocytochrome c, Smac/DIABLO, and Omi/HtrA2 are released rapidly following trophic factor deprivation in PC12 cells. Bcl-2 and Akt inhibited this release. The protection, however, persisted longer in differentiated PC12 cells. In differentiated, but not undifferentiated cells, Bcl-2 and Akt also inhibited apoptosis downstream of holocytochrome c release. Thus, undifferentiated PC12 cells showed marked sensitivity to induction of apoptosis by microinjected cytochrome c even in the presence of NGF, Bcl-2, or Akt. In contrast, in differentiated cells these factors suppressed cell death. Consistent with these observations, in vitro processing of procaspase 9 in response to cytochrome c was observed in extracts from undifferentiated but not differentiated cells expressing Akt or Bcl-2. Endogenous caspase 9 was cleaved during cell death, whereas dominant negative caspase 9 inhibited cell death. The results from determining the role of inhibitors of apoptosis (IAPs) suggest that acquisition of inhibition by IAPs is part of the differentiation program. Ubiquitin-DeltaN-AVPI Smac/DIABLO induced cell death in differentiated cells only. c-IAP-2 is unregulated in differentiated cells, whereas X-linked IAP levels decreased in these cells coincident with cell death. Moreover, expressing X-linked IAP rendered undifferentiated cells resistant to microinjected cytochrome c. Overall, the inhibitory regulation, of cell death at the level of release of mitochondrial apoptogenic factors and at post-mitochondrial activation of caspase 9 observed in differentiated PC12 cells, is reduced or absent in the undifferentiated counterparts.

Animals↗

PubMed2004

Macrophage-derived tumor necrosis factor alpha, an early developmental signal for motoneuron death.

Mechanisms inducing neuronal death at defined times during embryogenesis remain enigmatic. We show in explants that a developmental switch occurs between embryonic day 12 (E12) and E13 in rats that is 72-48 hr before programmed cell death. Half the motoneurons isolated from peripheral tissues at E12 escape programmed cell death, whereas 90% of motoneurons isolated at E13 enter a death program. The surrounding somite commits E12 motoneurons to death. This effect requires macrophage cells, is mimicked by tumor necrosis factor alpha (TNFalpha), and is inhibited by anti-TNFalpha antibodies. In vivo, TNFalpha is detected within somite macrophages, and TNF receptor 1 (TNFR1) is detected within motoneurons precisely between E12 and E13. Although motoneuron cell death occurs normally in TNFalpha-/- mice, this process is significantly reduced in explants from TNFalpha-/- and TNFR1-/- mice. Thus, embryonic motoneurons acquire the competence to die, before the onset of programmed cell death, from extrinsic signals such as macrophage-derived TNFalpha

Animals↗

PubMed2004

Intracellular association of glycine receptor with gephyrin increases its plasma membrane accumulation rate.

Gephyrin, a tubulin-binding protein, is the core of inhibitory postsynaptic scaffolds stabilizing glycine receptors (GlyRs) and/or GABA(A) receptors. Previous ultrastructural studies in vivo and in vitro have reported a localization of gephyrin to intracellular cisternas during development or after glycinergic denervation (Seitanidou et al., 1992; Colin et al., 1996, 1998). These data were compatible with a traffic of this cytoplasmic, but membrane-associated, protein together with membrane proteins such as GlyR after exocytosis and/or endocytosis pathways. We have now investigated the consequences of a GlyR-gephyrin interaction on the localization and the dynamics of these two molecules in African green monkey kidney cells (COS-7) cells and in neurons transfected with green fluorescent protein-tagged-gephyrin and myc-tagged GlyR alpha1 subunits. In these experiments, myc-tagged GlyR alpha1 contained, or did not contain, the gephyrin-binding sequence (betagb) of the GlyR beta subunit. We report here that GlyR-gephyrin interaction localizes gephyrin to GlyR-containing organelles. Videomicroscopy and nocodazole treatment indicate that the movements of these vesicles are microtubule dependent. Expressing GlyR alpha1 with a thrombin cleavage site between the myc-tag and the N terminal of the GlyR alpha1 subunit (Rosenberg et al., 2001) allowed monitoring of newly inserted receptors in the cell surface. Using temperature changes to block GlyR in, and then release it from, the trans-Golgi network, we show that gephyrin accelerates the accumulation of GlyR at the cell surface. Therefore, our data strongly suggest that some GlyR clusters are associated with gephyrin on their way to the cell surface and that this association increases the accumulation of GlyR at the plasma membrane.

Animals↗

PubMed2004

Morphologically identified glycinergic synapses in the hippocampus.

Inhibitory transmission in the hippocampus is predominantly GABAergic, but electrophysiological data evidenced strychnine-sensitive glycine-induced currents. However, synaptic currents have not been reported. Here, we describe, for the first time, the presence of GlyR clusters in several areas of the hippocampus as well as in cultured hippocampal neurons. In contrast with spinal cord, hippocampal GlyRs contain alpha2 but no alpha1 subunit. Optical and electron microscopy indicates that GlyRs can be synaptic as well as extrasynaptic. Synaptic GlyRs were apposed to glycinergic boutons characterized by the expression of the vesicular and the plasma membrane transporters of glycine (VIAAT and GlyT2, respectively). Double labeling with calcium-binding proteins showed that GlyT2 could be detected in boutons innervating both excitatory cells (soma and dendrites) and interneurons. Finally, GlyR clusters could be detected at synaptic sites with the GABAA receptor gamma2 subunit and gephyrin, suggesting that mixed GABA/glycine synapses might exist in the hippocampus.

Amino Acid Transport Systems↗