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    Title: 蛋白激脢之細胞訊息傳遞路徑中DARPP-32蛋白所扮演的角色：對大鼠學習記憶和抗細胞凋亡機制的影響
    Other Titles: Role and Mechanism of Darpp32 in Protein Kinase Ck2-Mediated Signaling Pathway: Influence on Learning &Amp;Memory and Anti-Apoptosis
    Authors: 趙知章
    Contributors: 國立政治大學神經科學研究所
    行政院國家科學委員會
    Keywords: 基礎醫學;生物技術;蛋白激脢;細胞訊息傳遞路徑;DARPP-32蛋白;大鼠;學習記憶;抗細胞凋亡
    Date: 2011
    Issue Date: 2012-11-15 11:23:49 (UTC+8)
    Abstract: 蛋白激酶 CK2 是屬於一種serine/threonine 類的蛋白激酶，目前已知會被CK2 磷酸 化的細胞受質分子數目已超過300 種以上，這些分子分別參與在傳遞細胞訊息、蛋白質 合成、細胞骨架結構、調節基因表現等等的機制之中。目前雖然已有一些在細胞分子生 物學層次的研究探討CK2 與促進神經可塑性的相關性，但是鮮少有探討CK2 參與神經 可塑性對於動物行為的影響；相較於神經可塑性的議題，CK2 與抗細胞凋亡相關的議題 則被廣泛探討，這些研究報告指出CK2 可以經由活化Akt/PKB、NF-κB 和ARC 蛋白 （apoptosis repressor with caspase recruitment domain）等機制來促進細胞的存活。蛋白 dopamine and cyclic AMP-regulated phosphoprotein, Mr. 32kDa（DARPP-32）最初是在紋 狀體腦區中被發現的，主要受到來自多巴胺的刺激增加了細胞內cAMP 的含量進而活化 Protein kinase A 對DARPP-32 蛋白進行磷酸化作用，除此之外，細胞內其他一些激酶如 CK2、CK1 和Cdk5 也會對DARPP-32 進行磷酸化作用，近年來的研究指出DARPP-32 除了與藥物成癮的生理現象有關外，應該還參與在其他生理調控或改變的機制之中，諸 如：DARPP-32 被發現與學習記憶歷程有著關聯性，但是並未討論到DARPP-32 蛋白上 已知可以被磷酸化的特定胺基酸與學習記憶之間的關連性；另外，DARPP-32 和其 isoform tDARPP 也在腸胃癌細胞內被發現有較高的表現量而且可以減緩癌細胞的死 亡，因此DARPP-32 也有參與在抗細胞凋亡機制之中的可能性。雖然根據DARPP-32 在 紋狀體腦區細胞內訊息傳遞過程中所擔負之功能的研究顯示，DARPP-32 應是扮演將來 自上游的訊息傳遞給其下游的protein phosphatase-1（PP-1），經由抑制PP-1 活性間接來 影響改變細胞的生理狀態，但是利用yeast two-hybrid 的研究技術發現在乳癌細胞中 DARPP-32 會與Discoidin Domain Receptor 1（DDR1）相結合，因此，DARPP-32 在神 經細胞內是否具有和特定蛋白結合的特性也是值得去探討的。由於DARPP-32 是CK2 磷酸化作用的其中一個受質，而在我們早些的研究中發現抑制CK2 的活性可以增進大 鼠對莫氏水迷津的學習記憶效果，我們最近的研究也發現CK2 參與在BDNF 抗細胞凋 亡的機制之中，而且也有文獻指出腦部損傷所引起的神經細胞凋亡是影響學習記憶的其 中一個原因，因此，承續我們上述的兩個研究結果，本研究計畫擬以活體實驗大鼠海馬 迴腦區為研究對象，探討DARPP-32 蛋白在CK2 調控的細胞訊息傳遞路徑中對學習記 憶和抗細胞凋亡的影響並且進行細胞機制上的探討，計畫擬定三年執行完成，各年計畫 目的如下：第一年研究重點是針對學習記憶的影響，包含（1）在活體實驗大鼠海馬迴 表 C011 共 2 頁第 1 頁 腦區確立CK2/DARPP-32/CREB 訊息傳遞路徑的相關性和（2）在活體實驗大鼠海馬迴 腦部調控CK2 活性或DARPP-32 表現是否會影響CREB 蛋白磷酸化和大鼠對於水迷津 的空間學習記憶；第二年研究重點針對神經細胞凋亡的影響，包含（1）在活體實驗大 鼠海馬迴腦區確立CK2/DARPP-32/Bcl-xL 訊息傳遞路徑的相關性和（2）調控CK2 活性 或DARPP-32 表現是否可拮抗興奮性神經毒性物質對神經細胞的傷害，以及在腦部損傷 狀況下提升抗細胞凋亡機制對學習記憶的影響；第三年研究重點則著重篩選細胞訊息傳 遞路徑中DARPP-32 蛋白特定的下游分子，利用DARPP-32 siRNA 和cDNA microarray 技術進行篩選（1）空間學習記憶機制中DARPP-32-mediated 的下游分子和（2）抗神經 細胞凋亡機制中DARPP-32- mediated 的下游分子。預期計畫的結果將有助於釐清 DARPP-32 蛋白在（1）學習記憶和抗細胞凋亡兩種不同細胞功能中的角色；（2）在 CK2-mediated 學習記憶和抗細胞凋亡機制的細胞訊息傳遞路徑中所扮演的角色；（3）有 哪些DARPP-32 的特定下流分子分別參與學習記憶和抗細胞凋亡的機制。 Protein kinase CK2 is a multifunctional and ubiquitous serine/threonine protein kinase. There are more than 300 substrates for CK2 that are involved in signal transduction, cytoskeleton structure, cell-cell adhesion and gene expression. Although there are some reports revealed that CK2 could promote the neuronal plasticity in cellular or molecular events, seldom of those reflects the relationship toward the behaviors regarding learning and memory. Beside the issue of neuronal plasticity, many studies of CK2 are focused on its anti-apoptotic effects. There are many reports showing that CK2 could activate protein kinase Akt, NF-κB and ARC protein (apoptosis repressor with caspase recruitment domain) and to enhance cell survival. Meanwhile, the dopamine and cyclic AMP-regulated phosphoprotein, Mr. 32kDa (DARPP-32) was originally found to be activated by dopamine and cAMP through PKA-dependent phosphorylation in the striatum. DARPP-32 was also found to be phosphorylated by other kinase such as CK2, Ck1 and Cdk5. Beside it well-known role in drug addiction, recent studies indicated that DARPP-32 might also involve in other physiological functions. The increase in DARPP-32 protein was found to parallel with the process of learning and memory. However, the relationship between DARPP-32 phosphorylation status and learning & memory hasn’t been investigated yet. Further, the higher protein expression of DARPP-32 and its isoform tDARPP were found in the gastrointestinal carcinomas and were relative to maintain the cell survival. The DARPP-32 was considered to indirectly influence the cellular functions through inhibition of protein phosphatase-1 in the striatum. However, the recent study revealed that DARPP-32 could interact with the Discoidin Domain Receptor 1 in the yeast two-hybrid system. This finding increases the possibility that the specific DARPP-32-mediated downstream molecules might exist in different cellular functions. In our past study, we found that the inhibition of CK2 could increase the performance of spatial memory in rats. We also found that CK2 activation was involved in the anti-apoptotic effects of BDNF in the recent study. There are some reports also revealed that brain injury-induced apoptosis would influence the performance of learning and memory. Since the DARPP-32 is one of substrates for CK2 phosphorylation, the present study is aimed to investigate the role of and mechanism of DARPP-32 involved in the CK2-mediated signaling pathway, specially focused on the influence on learning & memory and anti-apoptotic effect in the further three years. The specific aims of the first year are as following: (1) to investigate whether the relationships between CK2, DARPP-32 and CREB exist in the hippocampal CA1 regions of rats during the process of learning and memory; (2) to clarify whether manipulation of CK2 activity and DARPP-32 phosphorylation status will 表 C011 共 2 頁第 1 頁 influence the performance of learning and memory. The specific aims of the second year are as following: (1) to investigate whether the relationships between CK2, DARPP-32 and Bcl-xL exist in the hippocampal CA1 regions of rats in the anti-apoptotic mechanism; (2) to clarify whether manipulation of CK2 activity and DARPP-32 phosphorylation status will influence the anti-apoptotic effects against excitotoxicity and whether the increase in anti-apoptosis will improve the learning and memory impairment under neuronal damage. The specific aims of the third year are as following: by using the DARPP-32 siRNA followed by cDNA microarray techniques to screen and identify (1) the DARR-32-mediated downstream molecules during the process of learning and memory; and (2) the DARR-32-mediated downstream molecules in the anti-apoptotic mechanism. The expected goals of this proposal are: (1) to clarify the influence of CK2 on DARPP-32 in different cellular functions; (2) to build up the CK2/DARPP-32/CREB and CK2/DARPP-32/ Bcl-xL signaling pathway underline the learning & memory and anti-apoptotic mechanisms of CK2, respectively; (3) to specify the DARR-32-mediated downstream molecules correlative its different cellular functions.
    Relation: 基礎研究
    學術補助
    研究期間:10008~ 10107
    研究經費: 1100仟元
    Data Type: report
    Appears in Collections:[Graduate Institute of Neuroscience] NSC Projects

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