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Title: | LMBD1蛋白質參與視黃酸誘導神經樹狀突脊形成之探討 Roles of LMBD1 protein in retinoic acid-induced dendritic spine formation |
Authors: | Chen-Yin Lin 林承瑩 |
Advisor: | 張明富 |
Keyword: | 樹狀突脊,神經突觸,視黃酸, dendritic spine,synapse,retinoic acid, |
Publication Year : | 2014 |
Degree: | 碩士 |
Abstract: | 在神經元生長分化的過程中,樹突上形成之樹狀突脊為一重要結構,為突觸形成之位置,負責突觸傳訊,傳遞電訊號。樹狀突脊為一樹突上的膜突起構造,可接受來自軸突的訊息,因此軸突稱作突觸前細胞,樹突稱作突觸後細胞,樹突狀脊的典型構造,具有明顯澎大的頭部構造以及細的頸部構造,由肌動蛋白組成之骨架來支撐,代表樹突狀脊形態上的變化主要是和內部肌動蛋白的聚合相關。其中Rho-family small GTPases包含了RhoA、Rac和Cdc42會調控肌動蛋白的聚合進而影響樹狀突脊的形成。Rho GTPases的活性則受到guanine nucleotide- exchange factors (GEFs)以及GTPase-activating proteins (GAPs)的調控,Rho GTPases在和GTP結合時處於活化態,當GTP被水解成GDP時處於非活化態。GEF可以使Rho GTPases活化,GAP則是使Rho GTPases失活,而當Rho GTPases處於活化態時再和下游的分子作用,促使細胞骨架產生變化及啟動特定基因的轉錄進而影響樹突狀脊的形態變化及突觸的產生。Rac1和Cdc42會促進樹突狀脊生成,維持樹突狀脊的形態,而RhoA的作用相反,RhoA會導致樹突狀脊收縮,降低樹突狀脊的密度,限制突觸產生。先前的研究指出Dock4會和Elmo2形成複合物作為Rac1 GTPase的GEF,活化Rac1,可以正向調控樹突狀脊的生成,Dock4屬於CZH-Rho GEF蛋白質家族,和一般含有DH domain的GEF不同。有許多研究指出當樹突狀脊的形態出現異常時跟許多神經疾病像是自閉症、精神分裂症相關。lmbrd1基因主要經轉錄並轉譯出二種蛋白質LMBD1以及NESI。LMBD1比較長有540個胺基酸殘基,NESI比較短有467個胺基酸殘基,兩者差異在N端。LMBD1主要分布的位置在細胞膜、內質網及溶酶體的膜上。先前研究報導過LMBD1可以協助維生素B12從溶酶體內釋放到細胞質,而由本實驗室發現LMBD1會參與胰島素受體的內吞作用,進而影響葡萄糖的吸收。NESI則位於核膜上,為研究D型肝炎病毒大型delta抗原核輸出路徑時首次發現之蛋白質。然而對於這二種蛋白質在人體內之生理功能還有許多未知。本研究中所探討的是膜上的LMBD1在神經分化的過程中所具有的功能。先前本實驗室的研究發現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。另外,也發現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、神經突觸和樹狀突脊密度均下降,暗示LMBD1參與樹狀突脊之形成進而影響突觸的穩定性。本論文利用人類大腦神經芽細胞瘤細胞株SHSY-5Y為模式系統,以retinoic acid ( RA )誘導分化,將神經系統樹突狀脊的形成機制作為主要的研究目標。實驗結果顯示和沒有以RA誘導分化的細胞相比,利用RA誘導細胞分化後,發現LMBD1之表現量在神經細胞分化過程中表現量會下降隨後上升。同時發現當細胞內LMBD1表現量下降時,和野生型相比樹狀突脊的生成數量減少約70%,另外由免疫螢光分析結果顯示LMBD1和Dock4、Elmo2、Rac1在神經細胞分化的過程中有部份共位的現象,且在分化後期發現LMBD1和Dock4、Rac1於樹狀突脊位置上有明顯之共位現象,另外觀察到filopodia上有Elmo2的表現,LMBD1則主要表現在樹狀突脊生成之根部,推論Elmo2可能參與了filopodia之形成,間接促使spine之產生。接著進一步利用GST-pull down找到LMBD1上和Dock4、Elmo2、Rac1結合之不同區域,推論LMBD1與Dock4及Elmo2形成複合體去活化Rac1路徑,結果導致正向調控樹突狀脊之形成。 In neuronal development, the formation and growth of dendritic spines are important for the assembly of functional neuronal circuits. Dendritic spines are specialized actin-rich protrusions through which neurons receive excitatory inputs, representing the postsynaptic compartments of neuronal synapses. Dendritic spines typically consist of a spine head that is connected to the neuron by a thin spine neck. Filamentous actin is highly concentrated in dendritic spines, and spine morphogenesis is related to actin dynamics. The actin cytoskeleton is regulated largely by small GTPases, including Rho, Rac, and Cdc42. Dbl-homology (DH)-III domain-containing proteins are the classical guanine nucleotide exchange factors (GEFs) and that are responsible for activation of Rho GTPases. Activation of Rac1 and Cdc42 promotes spine formation, growth, and maintenance, whereas RhoA induces spine retraction and loss. It has been demonstrated that Dock4 forms a complex with Elmo2 to serve as a functional GEF for Rac1. Dock4 is a member of a newly discovered family, CZH protein, that can also act as Rho-GEFs. Indeed, spine abnormalities are associated with numerous neuropsychiatric disorders such as autism spectrum disorders, mental retardation, and schizophrenia. lmbrd1 gene mainly encodes two functional proteins NESI and LMBD1. The NESI protein participates in the nuclear export of the large hepatitis delta antigen with a proline-rich nuclear export signal. Whereas, the LMBD1 protein functions as a putative lysosomal exporter of vitamin B12 and a regulator of insulin receptor endocytosis. However, functions of the NESI and LMBD1 proteins 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 demonstrated that the densities of nerve fibers, synapses, and dendritic spines were lower in lmbrd1+/- mice. The results indicated that LMBD1 protein may have a role in spine formation. To further study the physiological roles of LMBD1 in nervous system, neuroblastoma SHSY-5Y cells were used to dissect its functions. In this study, the results showed that the expression level of LMBD1 was decreased within 24 hr after retinoic acid (RA) induction and then upraised at 72 hr and thereafter. In addition, knockdown of lmbd1 with shRNA impeded spine formation in SHSY-5Y cell line. Moreover, immunofluorescence staining indicated that LMBD1 colocalized with actin, Dock4, Elmo2, and Rac1 during RA-induced differentiation of SHSY-5Y cells, especially in spines at the late stage. The results indicated that Elmo2 may participate in filopodia formation, indirectly facilitating spine formation. Further studies demonstrated that different domains of LMBD1 were associated with Elmo2, Dock4, and Rac1 by GST- pull down assay. In conclusion, it is possible that LMBD1 forms complex with Elmo2 and Dock4 to activate Rac1 pathway, which, in turn, promotes spine formation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55936 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生物化學暨分子生物學科研究所 |
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