Recombinant myelin oligodendrocyte glycoprotein quality modifies evolution of experimental autoimmune encephalitis in macaques

The authors describe quantitatively and qualitatively different forms of experimental autoimmune encephalitis (EAE) in cynomolgus macaques. They found that bacterial contaminants within recombinant human myelin oligodendrocyte glycoprotein seemed to aggravate disease evolution. They provide anatomopathological features of fulminant and progressive forms of EAE, allowing them to distinguish specific factors influencing the evolution of this model of autoimmune demyelinating disease. Experimental autoimmune encephalitis (EAE) is a well-recognized model for the study of human acquired demyelinating diseases (ADD), a group of inflammatory disorders of the central nervous system (CNS) characterized by inflammation, myelin loss, and neurological impairment of variable severity. In rodents, EAE is typically induced by active immunization with a combination of myelin-derived antigen and a strong adjuvant as complete Freund's adjuvant (CFA), containing components of the mycobacterial wall, while myelin antigen alone or associated with other bacterial components, as lipopolysaccharides (LPS), often fails to induce EAE. In contrast to this, EAE can be efficiently induced in non-human primates by immunization with the recombinant human myelin oligodendrocyte glycoprotein (rhMOG), produced in Escherichia coli (E. coli), purified and formulated with incomplete Freund's adjuvant (IFA), which lacks bacterial elements. Here, we provide evidence indicating how trace amounts of bacterial contaminants within rhMOG may influence the course and severity of EAE in the cynomolgus macaque immunized with rhMOG/IFA. The residual amount of E. coli contaminants, as detected with mass spectrometry within rhMOG protein stocks, were found to significantly modulate the severity of clinical, radiological, and histologic hallmarks of EAE in macaques. Indeed, animals receiving the purest rhMOG showed milder disease severity, increased numbers of remissions, and reduced brain damage. Histologically, these animals presented a wider diversity of lesion types, including changes in normal-appearing white matter and prephagocytic lesions. Non-human primates EAE model with milder histologic lesions reflect more accurately ADD and permits to study of the pathogenesis of disease initiation and progression

Tissue-Specific Gain of RTK Signalling Uncovers Selective Cell Vulnerability during Embryogenesis

International audienceThe successive events that cells experience throughout development shape their intrinsic capacity to respond and integrate RTK inputs. Cellular responses to RTKs rely on different mechanisms of regulation that establish proper levels of RTK activation, define duration of RTK action, and exert quantitative/qualitative signalling outcomes. The extent to which cells are competent to deal with fluctuations in RTK signalling is incompletely understood. Here, we employ a genetic system to enhance RTK signalling in a tissue-specific manner. The chosen RTK is the hepatocyte growth factor (HGF) receptor Met, an appropriate model due to its pleiotropic requirement in distinct developmental events. Ubiquitously enhanced Met in Cre/loxP-based Rosa26 stopMet knock-in context (Del-R26 Met) reveals that most tissues are capable of buffering enhanced Met-RTK signalling thus avoiding perturbation of developmental programs. Nevertheless, this ubiquitous increase of Met does compromise selected programs such as myoblast migration. Using cell-type specific Cre drivers, we genetically showed that altered myoblast migration results from ectopic Met expression in limb mesenchyme rather than in migrating myoblasts themselves. qRT-PCR analyses show that ectopic Met in limbs causes molecular changes such as downregulation in the expression levels of Notum and Syndecan4, two known regulators of morphogen gradients. Molecular and functional studies revealed that ectopic Met expression in limb mesen-chyme does not alter HGF expression patterns and levels, but impairs HGF bioavailability. Together, our findings show that myoblasts, in which Met is endogenously expressed, are capable of buffering increased RTK levels, and identify mesenchymal cells as a cell type vulnerable to ectopic Met-RTK signalling. These results illustrate that embryonic cells are sensitive to alterations in the spatial distribution of RTK action, yet resilient to fluctuations in signalling levels of an RTK when occurring in its endogenous domain of activity

Comment le dialogue moléculaire entre bactéries et neurones change le comportement de l’hôte infecté

National audienceLes eucaryotes vivent dans un environnement contaminé par des microorganismes. Il n'est donc pas surprenant qu'ils aient forgé, au fil du temps, des relations extrêmement complexes et intimes entre eux. Les eucaryotes sont capables de percevoir la présence de bactéries et d’adapter leur réponse immunitaire, leur état physiologique ou même leur comportement en conséquence. Nombreuses sont les études qui ont démontré que les bactéries peuvent interagir avec le système nerveux eucaryote, soit au bénéfice du microbe qui modifie le comportement de l'hôte, soit au bénéfice de l'hôte qui adapte son comportement à l'infection. Dans la plupart des cas, cependant, les molécules et les mécanismes qui sous- tendent le dialogue entre les bactéries et leur système nerveux hôte n'ont pas été identifiés et leur mode d'action mal compris. Je présenterai les données obtenues avec le modèle Drosophile, sur la dissection des mécanismes cellulaires et moléculaires par lesquels un seul composé dérivé de microbiote, appelé peptidoglycane, influence le comportement des hôtes infectés en agissant directement sur certains neurones du cerveau

Deciphering the uranium target proteins in human dopaminergic SH-SY5Y cells

Uranium (U) is the heaviest naturally occurring element ubiquitously present in the Earth’s crust. Human exposure to low levels of U is, therefore, unavoidable. Recently, several studies have clearly pointed out that the brain is a sensitive target for U, but the mechanisms leading to the observed neurological alterations are not fully known. To deepen our knowledge of the biochemical disturbances resulting from U(VI) toxicity in neuronal cells, two complementary strategies were set up to identify the proteins that selectively bind U(VI) in human dopaminergic SH-SY5Y cells. The first strategy relies on the selective capture of proteins capable of binding U(VI), using immobilized metal affinity chromatography, and starting from lysates of cells grown in a U(VI)-free medium. The second strategy is based on the separation of U-enriched protein fractions by size-exclusion chromatography, starting from lysates of U(VI)-exposed cells. High-resolution mass spectrometry helped us to highlight 269 common proteins identified as the urano-proteome. They were further analyzed to characterize their cellular localization and biological functions. Four canonical pathways, related to the protein ubiquitination system, gluconeogenesis, glycolysis, and the actin cytoskeleton proteins, were particularly emphasized due to their high content of U(VI)-bound proteins. A semi-quantification was performed to concentrate on the ten most abundant proteins, whose physico-chemical characteristics were studied in particular depth. The selective interaction of U(VI) with these proteins is an initial element of proof of the possible metabolic effects of U(VI) on neuronal cells at the molecular level.Enhanced Eurotalents: a European programme for transnational mobility of experimented researchers managed by CE

Peptidoglycan-dependent NF-κB activation in a small subset of brain octopaminergic neurons controls female oviposition

Indexation en cours. PMCID: PMC6819134International audienceWhen facing microbes, animals engage in behaviors that lower the impact of the infection. We previously demonstrated that internal sensing of bacterial peptidoglycan reduces Drosophila female oviposition via NF-kB pathway activation in some neurons (Kurz et al., 2017). Although we showed that the neuromodulator octopamine is implicated, the identity of the involved neurons, as well as the physiological mechanism blocking egg-laying, remained unknown. In this study, we identified few ventral nerve cord and brain octopaminergic neurons expressing an NF-kB pathway component. We functionally demonstrated that NF-kB pathway activation in the brain, but not in the ventral nerve cord octopaminergic neurons, triggers an egg-laying drop in response to infection. Furthermore, we demonstrated via calcium imaging that the activity of these neurons can be directly modulated by peptidoglycan and that these cells do not control other octopamine-dependent behaviors such as female receptivity. This study shows that by sensing peptidoglycan and hence activating NF-kB cascade, a couple of brain neurons modulate a specific octopamine-dependent behavior to adapt female physiology status to their infectious state

A new procedure for high precision isotope ratio determinations of U, Cu and Zn at nanogram levels in cultured human cells: What are the limiting factors?

The monitoring of isotopic fractionations in in vitro cultured human cell samples is a very promising and under-exploited tool to help identify the metabolic processes leading to disease-induced isotopic fractionations or decipher metabolic pathways of toxic metals in these samples. One of the limitations is that the analytes are often present at small amounts, ranging from tens to hundreds of ng, thus making challenging low-uncertainty isotope ratio determinations. Here we present a new procedure for U, Cu and Zn purification and isotope ratio determinations in cultured human neuron-like cells exposed to natural U. A thorough study of the influence of the limiting factors impacting the uncertainty of δ238U, δ66Zn and δ65Cu is also carried out. These factors include the signal intensity, which determines the within-day measurement reproducibility, the procedural blank correction and the matrix effects, which determine the accuracy of the mass bias correction models. Given the small Cu and U amounts in the cell samples, 15–30 and 20 ng respectively, a highly efficient sample introduction system was employed in order to improve the analyte transport to the plasma and, hence, the signal intensity. With this device, the procedural blanks became the main uncertainty source of δ238U and δ65Cu values, accounting over 65% of the overall uncertainty. The matrix effects gave rise to inaccuracies in the mass bias correction models for samples finally dissolved in the minimal volumes required for the analysis, 100–150 µL, leading to biases for U and Cu. We will show how these biases can be cancelled out by dissolving the samples in volumes of at least 300 µL for Cu and 450 µL for U. Using our procedure, expanded uncertainties (k = 2) of around 0.35‰ for δ238U and 0.15‰ for δ66Zn and δ65Cu could be obtained. The analytical approach presented in this work is also applicable to other biological microsamples and can be extended to other elements and applications

Enhanced Met expression levels in <i>Del-R26</i>^<i>Met</i> myoblasts does not perturb activation of downstream signalling effectors.

<p>Limb transverse sections of E10.5 control and <i>Del-R26</i>^<i>Met</i> embryos showing the distribution of phospho-Met (on Tyr_1234–1235), phospho-Akt, phospho-ERKs (red) and of Pax3 protein (green) in myoblasts. Note ectopic phospho-Met in limb mesenchyme (arrowheads) and in non-migrating myoblasts (arrows) in <i>Del-R26</i>^<i>Met</i> mutants. Asterisks indicate non-specific staining in blood cells. Scale: 100μm.</p

Ubiquitously excess wild-type Met in developing embryos results into hyperflexed forelimbs.

<p>(A) Top: <i>Del-R26</i>^<i>Met</i> and control P0 mice showing hyperflexed limbs in mutants. Bottom: anti-myosin heavy chain II IHC using MF20 antibodies on forelimb transversal sections of P0 <i>Del-R26</i>^<i>Met</i> and control mice at the level of the forearm showing almost absence of extensor (asterisk) and a great reduction of flexor (arrowhead) muscle mass in mutants. (B, C) Whole mount ISH with <i>MyoD</i> probe of E12.5 embryos (B) and β-galactosidase staining of E11.5 embryos (C) showing that developing appendicular muscles are reduced in <i>Del-R26</i>^<i>Met</i> embryos (limbs are outlined in panels). The arrowhead in bottom panel B indicates developing ventral limb muscles (flexor). Scale: 500μm.</p

Myoblast migration is impaired in <i>Del-R26</i>^<i>Met</i> mutants.

<p>(A, B) Whole mount ISH of E10.5 embryos with <i>Lbx1</i> (A) and <i>Pax3</i> (B) probes showing drastic reduction of migrating myoblasts towards the developing tongue (arrowhead), fore and hind limbs. Bottom panel reports an enlargement at forelimb levels. (C, D) Quantification analyses of <i>Lbx1</i> (C) and <i>Pax3</i> (D) positive domains in forelimbs. Left panels: each plot represents the average signal distribution along the white line in forelimbs. Right panels: quantifications and statistical analyses of the sum of signal intensity based on intensity plots in left panels. Numbers of samples for <i>Lbx1</i>: control, n = 13; <i>Del-R26</i>^<i>Met</i>, n = 4; for <i>Pax3</i>: control, n = 11; <i>Del-R26</i>^<i>Met</i>, n = 8. The sum of <i>Pax3</i> signal intensity was calculated between point A and B: A indicating a fixed position between the somites and the limb whereas B being placed at a fixed distance from A. Note almost lack of signal in <i>Del-R26</i>^<i>Met</i> mutants. Scale: 500μm. Mann-Whitney and Student-<i>t</i> test.</p