Le FORUM, Vol. 39 No. 2

https://digitalcommons.library.umaine.edu/francoamericain_forum/1045/thumbnail.jp

Protein 4.1B Contributes to the Organization of Peripheral Myelinated Axons

Neurons are characterized by extremely long axons. This exceptional cell shape is likely to depend on multiple factors including interactions between the cytoskeleton and membrane proteins. In many cell types, members of the protein 4.1 family play an important role in tethering the cortical actin-spectrin cytoskeleton to the plasma membrane. Protein 4.1B is localized in myelinated axons, enriched in paranodal and juxtaparanodal regions, and also all along the internodes, but not at nodes of Ranvier where are localized the voltage-dependent sodium channels responsible for action potential propagation. To shed light on the role of protein 4.1B in the general organization of myelinated peripheral axons, we studied 4.1B knockout mice. These mice displayed a mildly impaired gait and motility. Whereas nodes were unaffected, the distribution of Caspr/paranodin, which anchors 4.1B to the membrane, was disorganized in paranodal regions and its levels were decreased. In juxtaparanodes, the enrichment of Caspr2, which also interacts with 4.1B, and of the associated TAG-1 and Kv1.1, was absent in mutant mice, whereas their levels were unaltered. Ultrastructural abnormalities were observed both at paranodes and juxtaparanodes. Axon calibers were slightly diminished in phrenic nerves and preterminal motor axons were dysmorphic in skeletal muscle. βII spectrin enrichment was decreased along the axolemma. Electrophysiological recordings at 3 post-natal weeks showed the occurrence of spontaneous and evoked repetitive activity indicating neuronal hyperexcitability, without change in conduction velocity. Thus, our results show that in myelinated axons 4.1B contributes to the stabilization of membrane proteins at paranodes, to the clustering of juxtaparanodal proteins, and to the regulation of the internodal axon caliber

Caractérisation fonctionnelle de récepteurs olfactifs de mammifères exprimés en lignées hétérologues (étude de l'effet de neuromodulateurs sur des cellules d'épithélium olfactif en culture primaire)

L'olfaction est un sens primordial qui a une influence importante sur le comportement des animaux. Bien qu'étudié depuis plus d'un siècle c'est seulement depuis un quinzaine d'années que les principaux acteurs moléculaires de la détection des odeurs, notamment les récepteurs olfactifs, ont été identifiés. Malgré cette découverte, les mécanismes moléculaires qui régissent la perception et la signalisation olfactive sont encore largement énigmatiques. Dans le travail qui a mené à cette thèse, j'ai développé un système d'expression hétérologue ou pseudo-homologue en cellules eucaryotes pour caractériser la réponse fonctionnelle de récepteurs olfactifs aux odorants. L'enjeu était de suivre les mécanismes de la détection et de la signalisation olfactive et d'envisager la possibilité d'un criblage des couples récepteurs olfactifs - odorants. Deux récepteurs olfactifs de mammifères, de ligands préférentiels déjà connus ont été utilisés pour développer les méthodes et initier l'étude, le récepteur 17 de rat et le récepteur 17-40 humain. Trois choix prépondérants ont été faits. D'une part, un système "minimaliste" d'expression de récepteurs "intègres", natifs (ou sans modification de leur séquence primaire), avec seulement une "étiquette" c-myc, a été utilisé pour perturber au minimum la reconnaissance du ligand et l'initiation de la signalisation cellulaire. Par contre, les systèmes d'expression ont été multipliés pour déterminer leur influence sur le niveau d'expression fonctionnelle. L'étude comparative a été effectuée sur des cellules de mammifères considérées comme "usines cellulaires" (COS, CHO, HEK), une lignée immortalisée issue d'épithélium olfactif de rat (adora). Enfin, la réponse fonctionnelle de clones stables ou de cellules exprimant transitoirement les récepteurs a été principalement étudiée par l'intermédiaire du calcium intracellulaire, second messager impliqué dans la transduction du signal olfactif, en spectrofluorimétrie ou en imagerie calcique. Par ailleurs, la mise au point d'un système de culture primaire de cellules de l'épithélium olfactif, s'est imposée en complément des systèmes d'expression hétérologue. Elle permettrait d'étudier d'une part les récepteurs olfactifs dans les cellules qui l'expriment naturellement, et d'autre part la modulation de ce signal olfactif par l'action de neuromodulateurs, dans le cadre de la régulation de l'olfaction par l'état physiologique, notamment nutritionnel. ...Olfaction is dramatically influencing animal behaviour. Despite one century of thorough investigations main molecular actors of the olfactory perception, namely olfactory receptors, have only been identified for a decade. Even with this important discovery, molecular mechanisms underlying olfactory perception and signalisation still remain unclear. During this PhD thesis I've set up a heterologous expression system in eukaryote cell lines in order to characterise a response of receptors to odorants by a functional approach. The aim was to follow mechanisms of olfactory detection and signalisation and to allow the screening of olfactory receptor/odorant pairs. Two olfactory receptors, namely the i7 rat receptor (rORi7) and the 17-40 human receptor (hOR17-40), which already have known ligands (octanal and helional, respectively), have been used in the study. Three leading choice have been made. First, the expression system was chosen as providing a native receptor protein expression with no modification within the intrinsic open reading frame (ORF) of the receptor (which, in the particular case of OR is always free of introns), accepting solely the addition of detection tag sequence (no secretion or import tag) like the c-myc sequence. We thus hoped to limit the possibly interfering events in ligand detection or signalisation triggering. The comparative study as been carried out on mammalian cell lines usually considered as cellular factory (CHO, COS or HEK cells) and on a conditionally immortalized cell line (ODORA) that was derived from the olfactory neuron precursor lineage: globose basal cells. Least, to achieved functional characterisation, I have chosen to focus on the intracellular calcium which is the main second messenger implicated in the olfactory transduction signal. Measurements were achieved using spectrofluorimetric and imaging techniques both in real time. Beside this approach, the use of olfactory cells primary culture quickly appeared as an important tools to complete the studies in heterologous systems. Primary culture of olfactory neurons could allow to study olfactory receptors in their natural hosting cells but also to look for the action of neuromodulators on the olfactory signal transduction. That second point would then offer the possibility of studying the influence of the animal physiological state, including nutritional state, on the olfactory capacity. ...ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Cometary dust properties retrieved from polarization observations: Application to C/1995 O1 Hale-Bopp and 1P/Halley

International audienceThe analysis of the polarized light scattered by cometary dust particles provides information on the physical properties of the solid component of cometary comae for C/1995 O1 Hale–Bopp and 1P/Halley. A model of light scattering by a size distribution of aggregates of up to 256 submicron-sized grains (spherical or spheroidal) mixed with single spheroidal particles has been developed, with its parameters adjusted to fit the phase angle and wavelength dependence of the polarization observations. The particles are built of two materials: a non-absorbing silicates-type material and a more absorbing organic-type material. The model reproduces accurately the inversion angle and the positive branch of the polarization phase curves from the visible to the near-infrared spectral domains. A negative branch of the polarization phase curves appears in our model, although the negative branch is not deep enough to reproduce accurately the observations. Significant differences are shown between the two comets, with dominance of small grains in the coma of Comet C/1995 O1 Hale–Bopp, well fitted by a distribution of the volume-equivalent diameter, a , following a−3.0 with a lower cutoff around 0.20 μm and an upper cutoff of at least 40 μm. For 1P/Halley, the size distribution follows a−2.8 with a lower cutoff around 0.26 μm and an upper cutoff of about 38 μm. The relative amount of organic-type particles is larger for 1P/Halley while the amount of aggregates, significant for both comets, is larger for C/1995 O1 Hale–Bopp

Laboratory light-scattering measurements with Titan's aerosols analogues produced by a dusty plasma

International audienceThe chemistry leading to the formation of solid aerosols (tholins) in Titan's atmosphere is simulated by a capacitively coupled plasma in a N2-CH4 gas mixture. The solid grains are produced in volume directly in the gas phase and studied ex-situ by SEM imaging and by light scattering on clouds of particles. The scattered light properties depend on the physical properties of the particles (morphologies, size distribution), as well as on the phase angle and the wavelength of the light. The particles may be aggregated or agglomerated grains. The grains size distribution is studied as a function of plasma parameters such as initial methane concentration introduced into the discharge, gas flow, absorbed RF power and plasma duration. The average grain size increases when the amount of CH4 increases, when the gas flow decreases, and when the plasma duration increases up to a limit for each production condition. For all the samples, the absorption decreases with increasing wavelength in the visible domain. As usually found for irregular particles, the polarization phase curves have a bell-shaped positive branch and a shallow negative branch. The maximum of polarization (Pmax) increases when the average grain size decreases (sub-μm-sized grains). To obtain Pmax values within the range of those measured in Titan's atmosphere; the average grains diameter has to be smaller than 100 nm, in agreement with the space observations results. In the light-scattering experiment, the size of the agglomerates in the clouds is in the 40-80 μm range in equivalent diameter. As a consequence Pmax increases with decreasing wavelength due to the increasing absorption, in agreement with observations of Titan from outside the atmosphere

Distinctive features of Egr transcription factor regulation and DNA binding activity in CA1 of the hippocampus in synaptic plasticity and consolidation and reconsolidation of fear memory.

International audienceActivity-dependent regulation of Egr1/Zif268, a transcription factor (TF) of the Egr family, is essential for stabilization of dentate gyrus synaptic plasticity and consolidation and reconsolidation of several forms of memory. The gene can be rapidly induced in selective brain circuits after certain types of learning or after recall. Here, we focused on area CA1 and examined regulation of Egr1, Egr2, and Egr3 mRNA and protein, and their DNA binding activity to the Egr response element (ERE) at different times after LTP in vivo and after learning and recall of a fear memory. We found LTP in CA1 leads to rapid induction of the three Egrs, however only Egr1 protein was overexpressed without a co-ordinated change in binding activity, indicating a fundamental difference between CA1 and dentate gyrus LTP. Our investigations in fear memory reveal that both learning and retrieval lead to an increase in binding of constitutively expressed Egr1 and Egr3 to the ERE, but not Egr2. Memory recall was also associated with increased Egr1 protein translation. The nature and temporal dynamics of these changes and tests for interactions between TFs suggest that in addition to ERE-mediated transcription, Egr1 in CA1 may interact with the TF c-Fos to regulate genes via other DNA response elements

Brain-Specific Basal and Novelty-Induced Alternations in PI3K-Akt and MAPK/ERK Signaling in a Middle-Aged AβPP/PS1 Mouse Model of Alzheimer's Disease.

International audienceAlthough it is well established that insulin/IGF and BDNF signaling are dysfunctionally regulated in Alzheimer's disease, there are very few studies documenting changes in major target proteins in different murine models of the disease. We investigated a panel of proteins in the PI3K-Akt and MAPK/ERK cascades in parietal cortex, dentate gyrus and CA1 in 13-month-old AβPP/PS1 transgenic mice to determine whether amyloid pathology is associated with basal dysregulation of these proteins or following exposure to novelty. The most striking effect we found was that there was little common regulation of proteins either by pathology alone or exposure to novelty across the three structures, suggesting dysfunctional mechanisms that occur simultaneously have important structure specificity. CA1 shared certain dysfunctional regulation of proteins in the MAPK/ERK cascade, but shared dysfunctional regulation of the PI3K/Akt cascade with the dentate gyrus. Changes in ERK/CREB in transgenic mice did not result in coordinated dysfunction of the downstream transcription factor, Egr1, as it was overexpressed in a normal manner following exposure to novelty. In the PI3K-Akt cascade, there was a flagrant increase in the levels of proteins associated with inflammation, such as NFκB, and structure specific regulation of proteins associated with autophagy, such as mTOR and FOXO1 and lack of regulation of Beclin-1. Finally, Beclin-1 was increased by novelty in wild-type mice but deficient in transgenic mice. Results are interpreted in terms of structure-specific dysfunctional regulation of signaling mechanisms associated with Alzheimer's disease

Light scattering by agglomerates: Interconnecting size and absorption effects (PROGRA2 experiment)

International audienc

Increasing endogenous glycine level prevents the induction of LTP.

<p><b>A:</b> Upper panel shows representative composite current responses of L5PyN for the range of imposed potentials before and during LTP. Scale bars: 150pA, 50ms. Medium panels displays the corresponding total conductance change gT before and during LTP. Lower panels show decomposition of gT into excitatory (gE, black) and inhibitory (gI, grey) conductances. Scale bars: 4nS, 50ms. <b>B:</b> Changes in IntgT up to 1 h post-TBS show that LTP is abolished when endogenous glycine levels are increased by blocking the glycine transporter with ALX (2μM) (n = 13 cells, 13 slices, 6 animals). The glycine degrading enzyme <i>Bs</i>GO has no effect on LTP (n = 19 cells, 19 slices, 9 animals), and prevents the effect of ALX (n = 13 cells, 13 slices, 6 animals), thus confirming that the latter is attributable to endogenous glycine rise. <b>C-D:</b> Excitatory (black) and inhibitory (grey) conductances were found to be equally affected by TBS application, regardless of the treatment, indicating that the E-I balance is unaltered by glycine and LTP. *p<0.05, **p<0.01, ***p<0.001 compared to pre-TBS.</p

D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period - Fig 2

<p>Glycine is not the endogenous co-agonist of L5PyNs VC NMDARs <b>A:</b> Application of the glycine degrading enzyme <i>Bs</i>GO (0.1 U/ml) has no effect on NMDA-EPSCs (n = 5 cells, 5 slices, 2 animals), indicating that glycine is not the endogenous co-agonist of synaptic L5PyrNs VC NMDARs. Scale bars: 100pA, 500ms. <b>B:</b> Further, enhancing endogenous glycine levels with the glycine transporter blocker ALX5407 (2 μM) decreased the NMDARs response, an effect blocked by <i>Bs</i>GO (n = 4 cells, 4 slices, 2 animals). Scale bars: 25pA, 250ms. <b>C:</b> A similar result is obtained by exogenous application of glycine (100μM) (n = 5 cells, 5 slices, 2 animals). Scale bars: 50pA, 500ms. <b>D:</b> Such downregulation of NMDA-EPSCs by glycine is remarkably blocked by the glycinergic receptors (GyRs) antagonist strychnine (10μM) (n = 5 cells, 5 slices, 2 animals). Scale bars: 50pA, 500ms. <b>E:</b> Immunofluorescence for GlyRs revealed that, in the VC, they are mainly expressed in L5PyRNs notably at the somatic and dendritic level. Scale bar: 50μm, inset: 30μm. <b>F:</b> Altogether, these results indicate that glycine downregulates NMDA-EPSCs through activation of GlyRs ** p<0.01, *** p<0.001.</p