Functional Analysis of LMBRD1 in Neuronal Differentiation

• Main Authors: Wei-Tse Hsu, 徐瑋澤
• Other Authors: Ming-Fu Chang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/29262488059689433121

碩士 === 臺灣大學 === 生物化學暨分子生物學研究所 === 98 === LMBRD1/NESI (nuclear export signal interacting protein), is a novel protein involved in the nuclear export of large hepatitis delta antigen. Previous studies from our laboratory demonstrated that LMBRD1 interacts with actin via α-actinin type actin binding domain. Knockdown of LMBRD1 resulted in increasing cellular mobility. In addition, heterozygous LMBRD1 knockout mice had defects of short term memory. The phenotypes of LMBRD1 (+/-) mice indicated that LMBRD1 may participate in the normal function of central nervous system. In this study, results indicated that LMBRD1 is highly expressed in mouse brain tissue and the expression level of LMBRD1 upraised after vascular epithelium growth factor (VEGF) induction. To further study the physiological roles of LMBRD1 in nervous system, neuroblastoma N2A cell model and primary culture systems were used to dissect function roles of LMBRD1. Knockdown of LMBRD1 with shRNA promoted neurite outgrowth and neuronal migration, but impeded neurite branching in both N2A cell line and primary culture. Moreover, immunohistofluorescence staining of specific neuronal markers demonstrated that the densities of nerve fibers, synapses, and dendritic spines were lower in LMBRD1 (+/-) mice. Results also demostrated that LMBRD1 colocalized with multiple cytoskeletal components, such as actin and β- tubulin, during differentiation of N2A cells. Moreover, LMBRD1 participates in specific molecular complexes of α-actinin, clathrin, AP2 and insulin receptor. Knockdown LMBRD1 interfered the binding of these components, suggesting a role of LMBRD1 in endocytosis. Immunostaining assay indicated that cellular distributions of clathirn and insulin receptor were changed in LMBRD1 knockdown N2A cells. The activity of insulin receptor was diminished in LMBRD1 knockdown N2A cells. Distribution of GluR2, a subunit of AMPA receptor, was retained on plasma membrane, suggesting that LMBRD1 could regulate AMPA receptor-mediated synaptic transmission. Results also suggested that these morphological phenotypes could be associated with elevated activity of Akt and cytoskeleton remodeling in neuron. However, the activity of Akt was not upregulated by insulin, suggesting that LMBRD1 may also involve in other signaling pathways.