Abstracts

Rationale: Defects in the development of connectivity underlie the pathogenesis of seizures and associated developmental intellectual disabilities. However, the molecular pathways involved in the achievement of normal connectivity are not completely understood. Here we use the animal model of X-linked lissencephaly, a mouse with a targeted deletion of the doublecortin (Dcx) gene conduct a proteonomic analysis of developing axon. The Dcx mutant mouse has spontaneously occurring seizures and recent work has shown that the doublecortin protein is a regulator of axonal transport. Thus, molecules important for axonal growth and connectivity may be deficient in the Dcx mutant axons. Methods: WT mice were fed with the 13C6-lysine diet starting at P20 and the bred. The F2 generation was completely labeled. The brains were immediately dissected after pefusion. Coronal brain sections were cut at 500 ?m using the Mcllwain Tissue Chopper. Corpus callosum or anterior commissure was isolated immediately from coronal brain sections and individually flash frozen. Unlabeled Dcx mutant axons will be combined with stable isotope labeled (SIL) WT samples. Proteins extracted from corpus callosum or anterior commissure of mouse embryonic brains are separated using gradient gel electrophoresis. Based on the staining, gels are sectioned and each band digested with trypsin and eluted. The resulting peptides were analyzed by liquid chromatography tandem mass spectrometry, with a nano-HPLC system connected to a hybrid mass spectrometer, the LTQ-Orbitrap-XL. Protein identification was performed with a search using the Sequest algorithm in the Bioworks Browser software against the Uniprot database. MS Spectra for each identified peptide will be extracted from raw data and Census, a quantification software will be used to determine the ratio of labeled vs. non-labeled peptides.Results: Initial analysis of samples from the anterior commissure of WT mice showed 1,376 proteins over. 99% of the proteins in the first run were also identified in the second run. 92% of the proteins found in the second run were found on the first run as well demonstrating consistency between two independent experiments. Of these proteins detected, a significant proportion (32%) were cytoskeletal, including microtubule, actin, motors and binding proteins. The other large category was for proteins with catalytic enzymatic activity- a heterogeneous collection of proteins. Finally, the presence of specific ion channels and of membrane receptors shows that this category of proteins is readily detectable in our samples demonstrating the feasibility of this technique for characterization of axon guidance and function.Conclusions: Quantitative mass spectrometry is feasible as a technique for conducting an unbiased screen on developing axons. Identification of defects in molecular pathways in mutant axons may highlight developmental systems critical for the pathogenesis of epilepsy.