Abstracts

One form of autosomal dominant partial epilepsy with auditory features (ADPEAF) is caused by mutations in the LGI1 (leucine-rich gene, glioma inactivated) gene, which was first identified as a putative tumor suppressor in glioblastoma cells. Apart from its decreased expression levels in these abnormally proliferating cells, little information is available concerning the normal function of the LGI/Epitempin protein in neurons, or how it causes an epileptic disorder affecting the temporal lobe. Furthermore, LGI1/Epitempin is only one member of a family of 4 highly related proteins. It is not known at this time whether the three other family members may also play a role in ADPEAF or, perhaps, in other forms of epilepsy. LGI1 contains a putative membrane spanning motif that may determine localization of the protein. In order to identify the functional pathway of this gene, we expressed LGI family members in various cell lines and localized the proteins by immunoblot techniques. cDNAs were generated by PCR from adult mouse brain. Epitope tagging (Flag (M2), hemagluttinin (HA), and myc (9E10)) was performed by PCR mutagenesis. 293T cells were transfected with cDNAs for LGI1 and additional family members. Both conditioned media and cell lysates were collected and analyzed by immunoblot. We observed that LGI1 is a constitutively secreted protein and find that approximately 90-95% of LGI1 protein is secreted with only a small fraction retained in the cell lysate, possibly in the ER, golgi and secretory vesicles. We have analyzed the additional LGI family members and find differences in cellular localization of the various family members that may be relevant to vesicle compartmentalization or could be due to epitope tagging which is necessary to visualize these other family members due to inavailability of specific antisera for LGI2-4. Our results strongly suggest that LGI1 is a secreted protein, thus having implications regarding CNS development. As a consequence of its secretion, LGI1 may exert its activities in a non-cell autonomous way, i.e. at some distance from the cell type in which it is initially produced. Further studies are underway to analyze the effects of LGI1 secretion on neuronal cell growth and survival in order to understand how LGI and its family members could contribute to an epilepsy phenotype. (Supported by NINDS NS29709, NINDS NRSA Training Grant NS045376-03, and Developmental Brain Disorder Training Grant NS43124)