Intitulé du sujet: Investigation of 24(S)-Saringosterol promoted sciatic nerve remyelination by Liver X Receptorβ Pathway in acute nerve injury mice

Sujet

Codirection: Julien Grenier et Charbel MASSAAD

Nombre de mois: 36 mois

Ecole Doctorale: ED 563 - Médicament,Toxicologie, Chimie, Imageries

Unité de recherche et équipe:

Unité de recherche et équipe（

The LXR Activity and Peripheral Nerve Remyelination research conducted at the Faculty of Basic and Biomedical Sciences of Université Paris Cité is internationally renowned. The laboratory facilities and advanced technologies at Université Paris Cité are at the forefront of the field. The laboratory T3S is located in the faculty of Basic and Biomedical Sciences that hosts technological platforms and facilities allowing to accomplish the project (Cytop2BM, dedicated to molecular biology and biochemistry i.e RT-qPCR, Nanodrop, WB, etc; Confocal imaging platform with LSM880 Zeiss Airyscan confocal microscope; Animal Facility and behavioral phenotyping facility). The UMRS1124 also provides cell culture, histology and cell biology facilities.It is of great significance to master the technical and theoretical research methods in order to explore the mechanisms of LXR activity and peripheral nerve remyelination.

The research group "Myelination and Nervous system pathologies" works on myelination and demyelinating diseases with a long-lasting expertise in the field of nuclear receptors, especially in LXRs. The team research is periodically supported by grants from the ANR, ARSEP (Association for MS), AFM (French association for myopathies), ERA-NET Neuron, IdEx, FRM, FRC and others.

Coordonnées de l’équipe:

INSERM UMR1124 UFR des sciences fondamentales et biomédicales, 45 rue des saints pères 75006 Paris

Secteur: Sciences de la vie / Life Sciences

Langue attendue: Anglais

Niveau de langue attendu: C2

Description

Description du sujet:

- Description   Neural demyelinating disease (demyelination) is a worldwide public health problem. At present, there are about 3-4 million patients worldwide, accounting for 6% -10% of the neurological morbidity[1-3]. Demyelinating disease is a kind of acquired disease with different etiology, different clinical manifestations, but similar characteristics. The pathological changes are characterized by the demyelination of nerve fibers and the relative integrity of nerve cells[4]. The role of myelin is to protect neurons and allow rapid transmission of nerve impulses on neurons[5]. Clinically, acute myelin injury resulting to demyelination is common.   The myelin sheath of the peripheral nervous system is formed by Schwann cells (SCs), which are multiple layers of specialized plasma membranes surrounding axons that facilitate efficient axonal signaling saltatory conduction and provide axons with the necessary nutritional support to maintain axonal integrity[6]. Myelination begins with the establishment of a 1:1 relationship with the axon. At this level, the production of myelin structural proteins such as myelin protein zero (P0), peripheral myelin protein (PMP22), myelin basic protein (MBP) are increased along with lipid biosynthesis[1, 7]. The peripheral nervous system exhibits a remarkable potential for repair and regeneration following acute nerve injury[8]. Successful repair and functional recovery rely largely on contingent Schwann Cells (SCs)[9]. SCs undergo a complex process of trans differentiation from mature SCs into a specific Repair SC phenotype that in turn re-differentiates into a remyelinating SCs[4]. In the past decade, extensive research has been directed in the direction of Schwann cells focusing on their physiological and neuroprotective effects on the neurons in the peripheral nervous system[9, 10].   In preliminary and unpublished experiments, Professor Charbel’s group identified an essential novel role for the liver X receptor β (LXRβ), a member of the nuclear receptor family of transcription factors, in early SC development[11]. LXRs exist in two different isoforms, LXRα or LXRβ. The natural ligands of LXRs are oxysterols originating from the oxidation of cholesterol by different enzymes [12-14]. LXR signaling has been implicated in several physiological processes such as lipid metabolism and cholesterol homeostasis in different systems including the central and peripheral nervous systems[15]. LXR implication in peripheral nerve repair focused little attention and was limited to its immunomodulatory effect[16]. In professor Charbel’s group previous studies, they demonstrate that the ablation of LXR signaling in full, constitutive LXRα/β knockout mice, impairs myelin integrity and nerve function via the Wnt/β-catenin pathway[17, 18]. Some researches point towards a novel and crucial role for glial LXRβ signaling in early peripheral nerve development and nerve integrity[19, 20].   A novel natural ligand of LXRβ has emerged, the 24(S)-Saringosterol exhibiting promising therapeutic advantages to treat nervous system pathologies among others[21, 22]. Studies has shown that 24 (S)-Saringosterol isolated from S. fusiforme can cross the blood–brain barrier and might exert modulatory effects on the central nervous system to cope with disorders such as AD[23]. Activation of liver X receptors (LXRs) by synthetic agonists was found to improve cognition in Alzheimer’s disease (AD) mice[24].   Thus, according to the preliminary study on LXR by Professor Charble group，I hypothesize that 24 (S) -Saringosterol has an important effects on SC state repair and myelin regeneration via Liver X Receptorβ pathway in sciatic nerve injuries. These investigations would contribute to a deeper understanding of LXRβ signaling in repair SC biology and greatly aid in assessing the true potential of LXR-based therapies to combat peripheral nerve disorders and ameliorating nerve regeneration following acute injury.     THE EXPERIMENTAL METHODS AND DATA ANALYSIS METHODS   1.Animal models The study will utilize 10 week old male and female LXRβ total KO mice(rossing CMV-Cre expressing mice with homozygous LXRβ-floxed individuals),their control counterparts (CMV-Cre expressing mice),and WT C57Bl/6 mice treated with 24(S)Saringosterol.All the groups will suffer an acute peripheral nerve injury realized by a unilateral crush lesion of the sciatic nerve.The Schwann cell injury response is conventionally studied for 4 consecutive weeks following an acute peripheral nerve injury realized by a unilateral crush lesion of the sciatic nerve.In all groups, nerve de- and regeneration will then be assessed at different time points after acute nerve injury (7, 14 and 28 days) on the behavioral, histological, and biological level.The elapsed time between the capture of a mouse and its death by decapitation was under 30 s. Sciatic nerves were collected and frozen in liquid nitrogen.   2.Effects of LXRβ on motor and sensory nerve function in acute nerve injury mice Automated gait analysis is a widely used method for evaluating functional recovery enrollment models of peripheral nerve injury, repair, and regeneration. The method can be used to study changes in gait after peripheral nerve injury, relating both to motor and sensory nerve function. During training sessions and on the test day, turn off all of the light sources in the behavioral test room and face the computer screen for data acquisition away from the camera to prevent its light from interfering with the camera. Bring the mice into the behavioral test room in their own home cage for at least 30 minutes before the test. On the first day of training, gently grasp an animal under its trunk and carefully place it in the walkway entrance.Let the rat explore the opening of the corridor without any interference, and wait until the animal voluntarily crosses the walkway to reach its home cage without any external stimulus or motivation. On the second day of training, allow the animals to become accustomed to entering the walkway and returning to their home cage without hesitation. On the third day of training, confirm that the animals have learned to cross the walkway at a uniform speed without hesitation, sniffing, or other explorative movements.On the fourth and fifth, repeat the training to reinforce the testing procedure. On the day of the experiment, use commercial glass cleaner and a squeegee to clean the top and bottom of the walkway taking care to remove all of the fluid from the ends of the walkway. For statistical analysis of the data, click view run statistics to obtain a comprehensive overview of the run statistics. Then select file and export, to export the run or trial statistics into a spreadsheet software.   3.Effects of LXRβ on nerve conduction velocity(NCV) and the compound muscle action potentials(CMAP) in acute nerve injury mice At 28 days post crush (28dpc), nerve function will be assessed by electrophysiological measurement of the nerve conduction velocity and the compound muscle action potentials.To measure NCV, mice were anesthetized with constant flow of isoflurane, and external body temperature was maintained at 34 °C with a heating lamp.For sciatic NCV, initial-ankle foot latency was measured using stimulating electrodes placed at the ankle and recording electrodes were placed dorsally over all five digits. The latency and distance between electrodes was measured, and then the stimulating electrodes were moved to the sciatic notch. The nerve was again stimulated and the resulting latency was subtracted from the initial ankle–foot latency. This difference was divided between the distance between the notch and ankle to determine velocity. The distance was determined by stretching the foot so that a linear distance could be measured between stimulating and recording electrodes.CMAP amplitudes were measured for each evoked response with electrode placement identical to the NCV measurements.   4.Effects of LXRβ on SC repair cells in acute nerve injury mice Electron microscopy will be utilized on 7 dpc to identify SC repair cells, which have a specific morphology and form regeneration clusters.Mice were deeply anesthetized and then intracardially perfused with 4% paraformaldehyde,and 0.1 m phosphate buffer, pH 7.4. Tissues were dissected and sciatic nerves were collected and immersed in the fixative solution at 4°C overnight, washed in phosphate buffer, postfixed in 2% osmium tetroxide, dehydrated in graded ethanol series, and embedded in epoxy resin. Semithin sections were cut with a glass knife at (0.5–1 μm) . For electron microscopy, ultrathin sections (50–90 nm) were cut on an ultramicrotome and collected on 300-mesh nickel grids. Staining was performed on drops of 4% aqueous uranyl acetate, followed by Reynolds's lead citrate. Ultrastructural analyses were performed in an electron microscope and digitalized with DigitalMicrograph software.The detailed structure of the Bungner band formed by SC repair cells was observed. Image acquisition was performed at the Imaging Facility. Electron microscopy images were used for calculating the g-ratio and axon perimeter using NIH ImageJ software.   Effects of LXRβ on generation and redifferentiation of the SC repair cell in acute nerve injury RNA and protein profiling of repair markers (Sox2, cJun, Shh, Olig1) at every time point(7dpc, 14 dpc and 28 dpc)to characterize the effect of LXRβ invalidation on the generation and redifferentiation of the SC repair cell. mRNA was analyzed by quantitative real-time PCR using the ΔΔCt method. Total RNA from mice sciatic nerves was obtained. One microgram was reverse transcribed with random primers and reverse transcriptase. PCR experiments were performed using TaqDNA polymerase and primers specific to each gene. PCR products were analyzed on agarose gel (2%) and visualized under UV.Quantitative real-time PCR was performed with standard protocols using SYBRGreen ROX Mix as a fluorescent detection dye in ABI PRISM 7000 in a final volume of 10 μl.To characterize the generated amplicons and to control the contamination by unspecific byproducts, a melting curve analysis was applied. Each reaction was performed in triplicate, and the mean of at least three independent experiments was calculated. Homogenates from sciatic nerves were prepared, and protein content was determined using the protein assay kit with BSA as standard. Western blot analysis was performed using rabbit polyclonal antibodies against and the appropriate secondary antibody, followed by exposure to radiographic film Hyperfilm ECL. Densitometric analysis was performed using National Institutes of Health ImageJ software. Oxysterols and cholesterol levels will be quantified by gas chromatography/mass spectrometry.Axonal regrowth will be assessed by immunohistochemistry with GAP43 antibody at 7 dpc.   Identify candidates of LXRβ modulation implicated in nerve repair and remyelination RNAseq experiments and bioinformatics analysis will be performed Identify candidates of LXRβ modulation implicated in nerve repair and remyelination. For RNA sequences, freshly dissociated SCs described were used . The RNA of SCs samples  were extracted using TRIzol and kit based on the manufacturer’s instructions. The integrity and quality of RNA extracts were assessed by an bioanalyzer, and samples of 1 μg or more with an RNA integ-rity number (RIN) of 7 or higher were used for RNAseq. Genes with P-value 2 were defined as differentially expressed genes. PCA plots were generated using the ggplot2 package in R with FPKM values.GO(Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genome) analyses were performed using the DAVID (https:// david. ncifc rf. gov/) database.The key signaling pathways will be obtained after GO and KEGG analysis of key genes and protein, then reverse transcription quantitative real-time polymerase chain reaction(RTq-PCR) and westernblotting.

Compétences requises:

Animal experimentation

Cell Biology 

Imaging

Molecular Biology

Références bibliographiques:

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