Vps18 In Murine Nerve Cell Survival, Migration And Dendritic Function And The Use Of PiggyBac Transposon Screening Tumor Suppressor Gene

Vps18gene encodes a protein of Vps C complex, which plays an important role in vesicle fusion at lysosome. However, its physiological function in mammals is unknown. We deleted Vps18in mice by gene targeting and found that Vps18-deficiency resulted in embryonic or early postnatal lethality. Then, we deleted Vps18specifically in neural cells by generating Vps18FI/FI; Nestin-Cre mice (Vps18CKO mice) and found that Vps18CKO mice displayed reduced body weight and died within12days after birth. Ablation of Vps18led to severe neurodegeneration, which was caused by blockage of multiple vesicle transport pathways to the lysosome, including autophagy, endocytosis and biosynthetic pathways. In Vps18-deficient neurons autophagosomes, late endosomes and multilaminar bodies were accumulated.We also found that mutation of Vps18led to impaired migration of neuronal cells and underdevelopment of dendrites of Purkinje cells. Our further study indicated that impaired neuronal migration might be attributed to disorganization of glial fibers and upregulation of β1Integrin in Vps18-deficient brain. Reducing the expression level of β1Integrin by RNAi could partially rescue the migration defect. Underdevelopment of Purkinje cell dendrite was likely resulted from the accumulation of Lox. Our results demonstrate important roles of Vps18in neuron survival, migration and dendrite development which are disrupted in multiple neural disorders in humans. Our research enhanced the understanding of neurodegenerative diseases and will facilitate the design of new therapeutic strategies for these diseases. Cancer is a leading cause of death worldwide. Despite decades of extensive scrutiny into the mechanism of tumorigenesis and metastasis, the battle against cancer is still far from over. A genome-wide screen for tumor suppressors will greatly enhance our understanding of the biology of cancer and facilitate the development of anti-cancer therapies.Retrovirus and transposons are powerful insertional mutagens. People have put strong promoters in them to carry out overexpression screens in the somatic cell of mice for oncogenes at the genome-wide level. However, becasuse of the low efficiency in using insertional mutagens to block both alleles of tumor suppressor in somatic cells, this system have had little success in the discovery of tumor suppressors. piggyBac (PB) transposon has been proved to be the most efficient transposon system in mammals. We generated a PB transposon to induce loss of function mutation in mice. This PB transposon have splicing accepter and polyadenylation signals inside and do not have strong promoter activity, therefore it will not activate the expression of surrounding genes after inserting into the genome. Using this PB transposon, we established transgenic mice with high PB copy number and high transposition efficiency. Combining this PB transposon with MMTV-neu breast cancer mouse models, we adapted the insertional mutagenesis system to screen for tumor suppressors. Our preliminary data showed that a PB transposon without strong promoter activity could not promote the initiation and metastasis of breast cancer in MMTV-neu mice, but induced heart tumor, which was never observed in control mice. By analyzing the PB insertion sites in heart tumors, we identified Ctnna3as a candidate tumor suppressor. Therefore, we proved for the first time that insertional mutagens can be used to screen for tumor suppressors in mice. Our work established the foundation to use PB transposon to carry our large scare screens for tumor suppressors in mice.