Alzheimer disease (AD) is a neurodegenerative disorder characterized by significant cognitive deficits, behavioral changes, sleep disorders and loss of functional autonomy. The number of patients suffering from AD is growing rapidly as the population ages worldwide. AD represents major cause of dementia in occidental countries. New revised AD diagnosis criteria were proposed. They highlighted the reduction of hippocampic volumetry by MRI, the reduction of glucidic metabolism in specific brain regions as well as alteration of CSF biomarkers: decrease of Aß1-42 peptides, increase of Tau and P-tau proteins. All these biomarkers help the clinical diagnosis with a sensitivity and specificity around 80% but the challenge of AD requires biomarkers beyond these probability and detectable in the blood. The use of news biomarkers in the field of the early diagnosis becomes a requirement because of the current development of therapeutic strategies aiming at slowing down, to even block the neurodegenerative process. Moreover, research in the field of biomarkers leads to relevant data to help elucidate Alzheimer pathophysiological mechanisms. One of the most interesting approaches is the analysis of inflammatory processes involved in AD. Incidentally, recent data suggested that AD pathological processes would produce disease-specific molecular changes in the blood, specifically on secreted signalling proteins, including cytokines, chemokines, and growth factors, as these are the primary means of communication between cells. Genomic investigation in human, as well as in AD animal models, also pointed out the implication of inflammatory molecules such as TNFs in the disease. We propose here further investigations on the involvement of inflammatory processes in AD. This approach is innovative in the field of blood biomarkers as it combines detection and identification of pro-inflammatory factors in human samples with dementia, and in transgenic animal models. A first part our project is based on the targeted and multiplex exploration and validation of inflammatory factors eventually identified in previous proteomic and genomic works. Both microfluidic and biochip multiplex technologies will be utilized. This will be done in patients (AD, prodromal AD, other dementia and inflammatory peripheral and central nervous system) and in a relevant AD transgenic mouse model: THY-Tau22. Data generated will be analysed with state-of-the art bioinformatics. We will then come full circle by cross-validating mouse and human models. In a second phase, putative biomarker candidates will be explored using immuno-histological approaches in mice, and will be measured in the CSF of patients. Our work program will therefore rationalize the identification and characterization of peripheral biomarkers of AD and help decipher the exact function of inflammation in neurodegeneration eventually leading to new therapeutic targets.

The project SmGKnhibit is aimed to the conception of novel and selective inhibitors of a specific ATP-dependent guanidino kinase of Schistosoma sp. (SmGK) a mammal parasite causing bilharzia diseases. - - The inhibitors will be designed to be selective against the parasite, by studying comparatively the host and parasite homologous enzymes and by exploiting specific differences identified in the enzymes. Detailed action mode of the prepared inhibitors, both at the molecular level as well as at the physiological level, will be provided in order to validate during the project SmGK as a potential new pharmacological target. - - We will take advantage of the newly developed fragment-based design methodology that we have recently developed in Lyon, France. The partners in Lyon have already collaborated for the validation of the chemical design methodology with thea report of novel and original inhibitor of mammal muscle creatine kinase (CK), the mammal homologue of SmCK [Bretonnet et al., J. Med. Chem. 2007 in the press). We have demonstrated that the methodology lead to high-value, novel hit molecules presenting original scaffold, simple enough to be easily developed by medicinal chemistry into more active and selective compounds. - - Our consortium includes an existing and validated in-house chemical library of molecular fragments suitable for initial affinity screening, a dedicated very high filed NMR system (600 MHz NMR and manipulating robots), high-performance molecular computation (in silico screening and interaction modelling), multi-step synthesis of designed potent inhibitors, evaluation of the enzymatic activity in vitro (partners 1, 2 CNRS - Université Lyon), then on cultured parasites and possibly on infected animals (partner 3, INSERM Pasteur INSERM Lille). - - All necessary prerequisite elements, as the parasite enzyme large-scale (10mg-scale) production as well as the structural biology basis are already available. The 3 partners are all leaders in their field of competences and own unic know-how and materials and dedicated instruments in France waranted the successness of the proposed project ...

Heart Failure (HF), especially secondary to myocardial infarction, still represents a major health concern worldwide. Improvement of HF treatment requires both to improve our knowledge on the molecular mechanisms of the disease and to development relevant biomarkers that may predict the outcome. Following this dual objective, both mechanistic and diagnostic, we initiated studies based on differential proteomics aimed at assessing, in a rat model of post-MI HF, the cardiac protein changes associated with chronic HF, and especially the post-translational modifications (PTM) of various contractile proteins, and at correlating these changes with the severity of adverse left ventricular (LV) remodeling and LV dysfunction. In parallel to the protein studies that allowed us to identify 27 differentially expressed proteins, we started studying PTM by assessing changes in phosphorylation and O-GlcNAcylation, two dynamic, reversible and often mutually exclusive processes involved in many cellular functions. We compared the phosphoproteome of LV in normal- and 2 month-HF rats using a combination of 2D electrophoresis and Pro-Q®Diamond staining. Among the 29 different phosphoproteins identified, we focused on proteins constituting the myofilaments :Troponin T (TnT), myosin light chain-3 (MLC-3), myosin light chain-2 (MLC-2), a1-tropomyosin (TMa-1), desmin and g-actin and the chaperone protein : aB-crystallin and Hsp 70. For each described proteins, we validated the modulation of phosphorylation using specific antibodies against the proteins and determined the aminoacid involved in the phosphorylation modulation. This analysis was pursued in more details with Tnt, for which the decrease in phosphorylation on serine208 revealed in the experimental model was confirmed in patients post IdM, and which forms the basis of a recent patent, and might present a major diagnostic value. Based on these promising results, our aim is now to better understand the role of these phosphorylation and O-GlCNAcylation processes in contractile cardiac defects in experimental HF. Several experimental models will be used: the model of MI induced by left coronary ligation, and a model of pressure overload hypertrophy (aortic stenosis), both in rats and mice, as well the isolated perfused heart (Langendorf), in order to correlate the PTM of the selected proteins, or their modulation, to the changes in cardiac contractility. For each protein, we will determine if they bear O-GlcNAc residues and in the positive case, we will determine if there is interplay between phosphorylation and O-GlcNAcylation in HF. We will characterize the enzymes involved in the phosphorylation (kinases and phosphatases) and O-GlcNAcylation (O-GlcNAc transferase (OGT) and O- GlcNAcase) interplay. Finally, concerning specifically Tnt, our production of specific antibodies for the phosphorylated form of the protein will allow to correlate the level of this phosphorylated form with the severity of HF, by tissue and cellular staining. This will help to study the role of PTM of TnT in the interaction with other myofilament proteins. The strength of the collaboration of the two groups is that they possess complementary expertise in experimental HF models and in proteomics. The former ANR project granted between the same two partners was fruitful with several publications and a patent.

Myotonic dystrophy type I (DM1) is a rare inherited neuromuscular disease affecting multiple organs. The genetic mutation is located on chromosome 19 and consists of dynamic expansion of a trinucleotide motif (CTG) in the 3’ untranslated region of DM protein kinase gene (DMPK). The aetiology of DM1 relies essentially on a toxic gain of function of RNAs transcribe from the mutated gene. Thus, RNAs bearing the long CUG expansion are retained in cell nuclei, associate to RNA-binding proteins, form proteoribonuclear inclusions named foci, ultimately leading to a defective splicing of several target transcripts. The defective splicing of each transcripts can be related to a specific symptom. Central nervous system involvement in DM1 was reported long ago, but systematic studies to analyze implicated brain structures, neuropathological hallmarks and molecular mechanisms, have only been initiated. We showed an altered splicing of RNA transcripts coding for a neuronal protein essential for maintaining the architecture and plasticity of neurons, the microtubule-associated Tau. This mis splicing is associated with the development of neurofibrillary degeneration. Neurofibrillary degeneration (NFD) is a neuropathological hallmark common to more than twenty neurological disorders named Tauopathies, to which DM1 belongs. Altogether, a growing body of evidences including our data suppose that a modified splicing of Tau is likely instrumental to NFD, and myotonic dystrophy is a good model to consolidate the proof of concept of the relationship between the indirect mis splicing of Tau, brain function and the development of NFD. Therefore, our global objective is to determine the contribution of a modified splicing of Tau to the development of NFD, to reverse the mis splicing of Tau and we intend to model, study and reverse this pathophysiological mechanism in novel transgenic mouse models of NFD in DM1. To that purpose, we propose to cross DM1 transgenic mice expressing the mutated DM1 human locus with mice transgenic for the human TAU gene but invalidated for the endogenous murine Tau gene. The resulting transgenic offspring will enable to address our hypothesis. Our preliminary results demonstrated an altered splicing of Tau, hyperphosphorylation of endogenous Tau and altered synaptic plasticity in the DM1 transgenic mouse model. However, the endogenous splicing of the murine Tau is different from that of the human TAU splicing pattern. In contrast, the human TAU transgenic model is expressing the six Tau isoforms alike in the human brain. By multidisciplinary approaches and complementary expertises of the two laboratories implicated in this project, we expect to develop and characterize DM1 x human TAU transgenic mouse model that will reproduce a NFD process similar to the human pathophysiology, associated with Tau splicing defects. Moreover, our preliminary results have established that Tau mis splicing directly result from the loss of function of a splicing factor that is MBNL. We have engineered a chimeric splicing factor that enables to correct the mis splicing of Tau in vitro. Moreover, in control condition, ectopic expression of this construct is not toxic and does not regulate the splicing of Tau and other targets. We are currently producing the lentiviral vector in order to infect DM1 mice as well as the future hTAU x DM1 transgenic models. Therefore, besides being essential to address fundamental questions, the DM1 model of NFD we will develop will be very useful to test drugs and to test viral gene transfer in order to rescue in vivo the mis splicing of tau and most likely the NFD and associated symptoms. Altogether, our project aim to develop a completely novel transgenic mouse model of NFD and to rescue the pathophysiology of DM1 using a viral gene transfer therapy.