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- 學位論文
LMBD1蛋白質參與神經突脊形成之作用機制
The Mechanism of LMBD1 Protein Involved in Neuronal Spine Formation
摘要
在神經元生長分化的過程中，樹突上形成之樹突狀脊為一重要結構，為突觸形成之位置，負責突觸傳訊，傳遞電訊號。lmbrd1基因可產生二種主要的蛋白質，分別是NESI以及LMBD1。其中LMBD1會參與胰島素受體的內吞作用，以及協助維生素B12從溶酶體內釋放到細胞質，然而對於lmbrd1基因在人體內之生理功能還有許多未知。先前本實驗室的研究發現lmbrd1+/-基因剔除小鼠在行為上有短期記憶的異常，暗示lmbrd1可能參與中樞神經系統正常功能之調控。另外，利用組織免疫螢光染色分析神經分子標記也發現在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，暗示LMBD1參與樹突狀脊之形成進而影響突觸的穩定性。為了更進一步地探究LMBD1在神經系統上所扮演的角色，實驗室利用人類大腦神經芽細胞瘤細胞株SH-SY5Y進行研究，發現以shRNA抑制LMBD1表現後，樹突狀脊的形成會受到抑制。免疫螢光分析結果顯示LMBD1和Dock4、Elmo2、Rac1在神經細胞分化後期於樹突狀脊位置上有明顯之共位現象。因此推論LMBD1可能與Dock4及Elmo2形成複合體去活化Rac1路徑，正向調控樹突狀脊之形成。在本研究中欲探討LMBD1透過何種機制調控樹突狀脊之形成，結果顯示LMBD1會透過其羧基端 (胺基酸383-540) 以及中間區域 (胺基酸286-383) 分別和Dock4以及Rac1結合。由於Dock4可作為GEF活化Rac1，因此本研究表現並純化TAT-LMBD1(386-540) 融合蛋白質，將其送入細胞，藉由競爭內生性的LMBD1檢視其功能。結果發現TAT-LMBD1(386-540) 融合蛋白質的存在會影響SH-SY5Y細胞株之分化，甚至是細胞之其他生理功能。此外，利用shRNA抑制lmbrd1基因表現時發現，Rac1之活性也同樣受到抑制。以上結果顯示LMBD1可能擔任支架蛋白質之角色，促使Dock4-Elmo2複合體移動到細胞膜附近，並且促進Rac1傳訊途徑，正向調控樹突狀脊之形成。
並列摘要
In neuronal development, the formation and growth of dendritic spines are critical for the assembly of functional neuronal circuits. Lmbrd1 gene encodes two major functional proteins, NESI and LMBD1. The LMBD1 protein functions as a regulator of insulin receptor endocytosis and a putative lysosomal exporter of vitamin B12. However, functions of the lmbrd1 gene in human cells are largely unknown. Our previous studies demonstrated that heterozygous lmbrd1 knockout (lmbrd1+/-) mice had defects of short term memory, indicating that lmbrd1 may participate in the normal function of central nervous system. In addition, immunohistofluorescence staining of specific neuronal markers in the hippocampus of lmbrd1+/- mice demonstrated decreases on the densities of nerve fibers, synapses, and dendritic spines. The results indicated that LMBD1 protein may have a role in spine formation. For further understanding the physiological roles of LMBD1 protein in nervous system, neuroblastoma SH-SY5Y cells were applied. Knockdown of lmbrd1 gene with shRNA impeded spine formation. In addition, LMBD1 protein colocalized with Dock4, Elmo2 and Rac1 during RA-induced differentiation of SH-SY5Y cells, especially in spines at the late stage. It is possible that LMBD1 forms complex with Elmo2 and Dock4 to active Rac1 pathway, and promotes spine formation. In this study, the specific aim is to investigate the mechamism of LMBD1 protein involved in neuronal spine formation. The results showed that LMBD1 interacted with Dock4 and Rac1 through its carboxyl-terminus and middle domain, respectively. Because Dock4 serves as a GEF to activate Rac1, a TAT-LMBD1(386-540) fusion protein was generated and used to interfere LMBD1 function through competition with endogenous LMBD1. The results indicated that the TAT-LMBD1(386-540) fusion protein impeded the differentiation, and even other physiological functions in SH-SY5Y cells. Furthermore, knockdown of lmbrd1 gene by shRNA attenuated Rac1 activity. The results suggest that LMBD1 acts as a scaffold protein to recruit Dock4-Elmo2 complex to the plasma membrane, which facilitates the signaling of Rac1 pathway and promotes the spine formation.