LMBRD1蛋白質對神經分化的功能性分析

Abstract

LMBRD1/NESI (nuclear export signal interacting protein)是一參與D型肝炎病毒大型delta抗原核輸出之新發現蛋白質。本實驗室先前的研究證明LMBRD1會透過其α-actinin類型肌動蛋白結合位與F- actin結合，且LMBRD1 knockdown會增加細胞移動能力。 此外，LMBRD1+/-基因剔除小鼠行為上顯示有短期記憶的異常，暗示LMBRD1可能參與中樞神經系統正常功能之調控。 在本論文中，實驗結果顯示LMBRD1高量表現於小鼠大腦組織中。在利用VEGF誘導神經纖維瘤細胞株N2A分化後，發現LMBRD1之表現量在神經細胞分化過程中隨時間而提高。為了進一步了解LMBRD1對神經分化之影響，利用N2A細胞株和小鼠神經元分離培養，分別證實在LMBRD1表現量較低時，神經突之延長以及細胞遷移速度較快，但神經突分支則減少。另外，我們也利用數種神經分子標記經組織免疫螢光染色證實在LMBRD1+/-小鼠海馬迴切片中，神經纖維、神經突觸和樹突狀脊密度均下降。實驗結果亦顯示LMBRD1與多種細胞骨架蛋白質在N2A細胞免疫螢光染色中共位，包含actin與β- tubulin。另外利用免疫沉降法證實α-actinin, clathrin, AP2與 insulin receptor (IR) 在細胞中與LMBRD1共同形成免疫結合體，且其結合強度受到LMBRD1表現量影響，顯示LMBRD1可能參與內吞作用之調控。免疫螢光染色亦顯示IR與clathrin之分布會受到LMBRD1表現量影響，而AMPA受體之次單元體GluR2亦會隨著LMBRD1表現量降低而聚集在膜上，顯示LMBRD1可能影響神經突觸之傳訊。另外我們也發現在LMBRD1 knockdown細胞中，Akt的活性上升，與先前觀察到的表徵可能有關，但其活性不受insulin所活化，這顯示可能有其他訊息傳遞路徑參與其中。

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.