DYNAMIC PROTEOME ANALYSIS OF ANTERIOR MICE HIPPOCAMPUS

Neurodegeneration is a molecular process that occurs during several brain diseases (Parkinson, Alzheimer, Epilepsies). This process leads to the reorganization of synapses and neurons in the Central Nervous System. The search for early medication to reduce neurodegeneration is a major challenge in Neuroscience. To this aim VALAPODYN, a European Commission-funded research network, develops functional genomics and proteomics related to the dynamics of molecular interaction networks (MIN). MIN modeling investigates protein-protein interactions and regulation networks. This study is established on scientific literature and on experimental data coming out of genomic and proteomic analyses. Experimental data are obtained thanks to a model of kainate inducing cell loss with focal epilepsy in mice hippocampi . This model permit the EU Consortium to get a transcriptome and proteome dynamic expression analysis conducted at 10 successive time-points during the first 24h after kainate induction. Proteome dynamic analysis is performed with total and fractionated proteins extracts. Obtained proteins are separated by two methods: (i) a classic method based on 2D-PAGE gel separation of these proteins; (ii) a method based on the 2D-HPLC liquid separation of digested peptides. When proteins are separated on 2D-PAGE, proteins spots are visualized thanks to 2 images analysis software’s using different mathematical algorithms. Proteins spots are picked and digested automatically using a spot picker and a recently acquired digester workstations. Mass spectrometry identification of digested peptides (2D PAGE MALDI-TOF/TOF and 2D-LC MS/MS) highlighted the modulation of the expression of 232 different proteins during the first 24h after kainate induction. The dynamic proteome analysis was performed thanks to complementary strategies to cover a large window of molecular events as a result of cellular loss induction. “User-friendly” documents were constructed to efficiently communicate proteome information. The obtained lists of proteins were extensively analysed with public databases (UniProtKB, NCBI, Pfam etc) to review interactions, biological functions, protein families with different isoforms and cellular-tissue localisation. We want also to emphasize that several proteins families showed multiple spots as a result of different isoforms. These isoforms are probably the consequence of natural chemical modifications. The study of these chemical modifications in the native conditions needs a dynamic analysis of the tri-dimensional structure and folding showed by these isoforms. This new study will permit to establish a link between proteomic, interactomic data and data issue from the structural biology.‘Validated Predictive Dynamic Model of Complex Intracellular Pathways Related to Cell Death and Survival