Date : 02/08/2012

Internship proposal for : Master 1 or Master 2

Laboratory
INSERM/UEVE UMR 861, I-STEM, AFM
UMR 861, INSERM
5, Rue Henri Desbruères
91030 Evry
Website
Main discipline : Cell Biology
Lab director : Marc Peschanski

Mentor
Cécile Martinat
email : Cet e-mail est protégé contre les robots collecteurs de mails, votre navigateur doit accepter le Javascript pour le voir
phone : +33 1 69 90 85 33

Subjects
1.: disease modeling
2.: human embryonic stem cells
3.: Myotonic dystrophy type 1

Tools and methodologies
1.: cell culture of human embryonic stem cells
2.: differentiation of human embryonic stem cells into motoneurons,microfluidic chamber
3.: immunofluorescence, western blot, qRT-PCR, live cell imaging

Summary of lab's interests
The intership will be done in Istem, an institute fully dedicated to the use of Human Pluripotent Stem Cells for the study and the treatment of monogenic diseases. Our team is interested in the use of disease-specific Human Pluripotent Stem cells to decipher physiopathological mechanisms implicated in the development of neuro-muscular diseases and subsequently in the identification of new therapeutic compounds through drug screening application. We evaluate the feasibility of this concept by using human embryonic stem cells (hESCs) carrying the causal mutation of the Myotonic Dystrophy Type 1 (DM1) which is the most common form of inherited muscular dystrophy in adults. DM1 is the most common form of inherited muscular dystrophy in adults. It involves various multisystemic clinical manifestations including myotonia (delayed muscle relaxation after voluntary contraction), progressive wasting of skeletal muscle, cardiac conduction defects and extramuscular symptoms such as cataracts, endocrine and nervous system impairments. This disease, due to an abnormal CTG expansion in the 3' untranslated region of the a gene called DMPK, results in the retention of the repeat-containing transcripts in the nucleus as ribonucleoproteins inclusions, sequestrating RNA-binding proteins leading to defects in different gene expression or alternate splices. Whereas pathological mechanisms of the muscular system are well described, less is known about the central nervous system. Our goal is to use DM1-hESCs to improve our knowledge on the pathological consequences of DM1 in the central nervous system.

Summary of project
By using DM1-hESCs derived motoneurons, we previously identified a defect in neuritogenesis and synaptogenesis that we correlated to an altered SLITRK expression. Interestingly, we showed that these functional defects could affect the interaction between motoneurons and their target muscle cells (Marteyn et al., 2011). Using the same cellular system, we recently identified a new genetic alteration affecting the alternate splice of the receptor to ephrinA5 (EphA5), a gene also known to be implicated in the neurotigenesis and synaptogenesis. Validating the pertinence of this finding, we confirmed EphA5 altered splice both in biopsies from DM1 patients and also in a DM1 murine model. Thus, in DM1 cells, we observed an increased inclusion of exon 9 of EphA5. This project will focus on two questions: 1. How does DM1 mutation affect EphA5 splice ? DM1 mutation affects the function of several RNA-binding proteins such as Mbnl1 and CUGBP1, known to be implicated in the control of splice events. By using a minigene containing the exon 9 of EphA5 and the adjacent intronic sequence, we will addressed whether overexpression or downexpression of Mbnl1 and CUGBP1 affect the inclusion of exon 9. 2. What are the functional consequences of the altered EpHA5 splice? The role of EphA5 on neuritogenesis is well described. However, nothing is known about the effect of the exon 9 on the function of this receptor. EphA5 exon 9 encodes for a small sequence located just underneath of the transmembrane domain. Predictive structural analyses suggest that exon 9 inclusion or not could modify the structure of the intracytoplasmic domain and subsequently modify the intracellular signaling. This hypothesis will be tested by using control and DM1-hESCs derived motoneurons treated with the ephrinA5 ligand and the cell signaling will be analyzed by western blot. The effect of the splice defect will be also analyzed on the neuritic outgrowth by using microfluidic chamber. This technique in association with live cell imaging and immunostaining will allow to specifically stimulating the neuritic outgrowth after local treatment with ephrinA5 ligand.