Inhibition of N1-Src kinase by a specific SH3 peptide ligand reveals a role for N1-Src in neurite elongation by L1-CAM

In the mammalian brain the ubiquitous tyrosine kinase, C-Src, undergoes splicing to insert short sequences in the SH3 domain to yield N1- and N2-Src. We and others have previously shown that the N-Srcs have altered substrate specificity and kinase activity compared to C-Src. However, the exact functions of the N-Srcs are unknown and it is likely that N-Src signalling events have been misattributed to C-Src because they cannot be distinguished by conventional Src inhibitors that target the kinase domain. By screening a peptide phage display library, we discovered a novel ligand (PDN1) that targets the unique SH3 domain of N1-Src and inhibits N1-Src in cells. In cultured neurons, PDN1 fused to a fluorescent protein inhibited neurite outgrowth, an effect that was mimicked by shRNA targeting the N1-Src microexon. PDN1 also inhibited L1-CAM-dependent neurite elongation in cerebellar granule neurons, a pathway previously shown to be disrupted in Src(−/−) mice. PDN1 therefore represents a novel tool for distinguishing the functions of N1-Src and C-Src in neurons and is a starting point for the development of a small molecule inhibitor of N1-Src

Telencephalic transplants in mice: characterization of growth and differentiation patterns.

Telencephalic grafting represents a powerful tool for developmental studies and for the investigation of biological features of transgenic brain tissue. The interpretation of grafting experiments, however, requires detailed knowledge of graft biology. Therefore, we have characterized growth rates, graft size, and differentiation of embryonic telencephalic tissue harvested at various developmental stages and grafted into the caudoputamen and lateral ventricles of histocompatible mice. A total of 164 grafts were analysed up to 500 days after transplantation. Of all transplants, 79.3% resulted in the formation of solid neural grafts. Grafted cells were identified by 3H-thymidine labelling and autoradiography. Proliferation was studied by bromodeoxyuridine incorporation and decreased from an initial 35% at 1-3 d after grafting to less than 1.6% after 40 days. The graft size was measured as a function of the embryonic age of the transplanted tissue. Our data indicate that telencephalic tissue harvested at embryonic day E 12.5 reproducibly yields large, fully differentiated neuroectodermal grafts. The parameters defined in this study will be useful for detailed analysis of neuroectodermal tissue from mice undergoing fatal neurodegeneration, such as knockout mice bearing lethal mutations