TP4: Proteomic analysis of the presynaptic active zone from APP/APLP mutant mice
Synaptic vesicles docked to the presynaptic plasma membrane comprise the exocytosis-competent subpopulation of vesicles at the active zone. Employing subcellular fractionation of synaptosomes derived from mouse brain and a monoclonal antibody directed against an integral synaptic vesicle membrane protein, we could immunoisolate synaptic vesicles together with the attached presynaptic plasma membrane. All three members of the APP family are present in the immunoisolated active zone fraction. Our goal is to compare the proteome of the presynaptic active zone of APP/APLP-knockout mice to that of controls. For this purpose different proteomic approaches will be applied, either gel-based, i.e. DIGE (differential gel electrophoresis) using dedicated gel techniques to promote the separation and detection of membrane proteins, or 2D-HPLC MS/MS techniques of the enzymatically generated peptide mixtures. Moreover, using these techniques we will investigate in addition if there are differences in the posttranslational modifications of presynaptic proteins following induced synaptic activity between littermate controls and APP/APLP mutant mice. Differences in the protein profile will be analyzed and the functional consequences evaluated.