- 學位論文
β-catenin在BCAS2條件式剔除小鼠及阿茲海默症疾病模式小鼠中調控成體神經新生過程之角色
The role of β-catenin in adult neurogenesis of BCAS2 cKO mice and Alzheimer’s disease model (APP) mice
摘要
BCAS2是一個26kDa大小的核蛋白，它是腫瘤抑制因子p53的負調控者，參與在細胞的生長調節，並且也是Prp19/CDC5L RNA剪切複合體的核心蛋白，為pre-mRNA剪切活動所必需。我們實驗室先前的研究發現BCAS2會參與調控β-catenin pre-mRNA 的剪切，進而影響β-catenin蛋白表現，在透過CaMKIIα驅動而產生BCAS2缺乏的BCAS2條件式基因剔除小鼠中，我們發現β-catenin蛋白表現有顯著降低，並且伴隨海馬迴中齒狀迴內新生神經樹突發育的異常及退化的學習能力與記憶。我們過去利用基因慢病毒載體攜帶shBCAS2對野生型小鼠進行BCAS2敲落，發現神經幹細胞的增生比率被顯著降低，而相對的，對BCAS2條件式基因剔除小鼠進行顱內注射AAV-BCAS2，我們發現神經幹細胞的增生以及β-catenin蛋白表現能夠恢復。由於已有許多報導指出Wnt/β-catenin訊息傳遞鏈的活化會促進神經幹細胞的自我更新以及刺激神經的分化，因此β-catenin普遍被認為在成體神經新生中扮演重要角色。而我們也因此認為BCAS2調控神經幹細胞的增生是透過β-catenin。在本篇研究中，我們首先探討是否DCX+新生神經的生長也會受到BCAS2影響，而結果顯示在BCAS2條件式剔除小鼠及BCAS2敲落小鼠中，DCX+細胞數量都有顯著降低。相對的，顱內注射AAV-BCAS2則回復了BCAS2條件式剔除小鼠中DCX+細胞數量，結果指出BCAS2確實也參與在神經分化的調控中。另外，為了探討BCAS2透過β-catenin調控成體神經新生的假設，我們設計利用AAV-β-catenin注射BCAS2條件式剔除小鼠以強行表現β-catenin的方式能否改善神經新生。結果透過神經幹細胞指標Sox2、DNA合成指標BrdU、未成熟神經細胞指標DCX及增生活動指標Ki67，顯示神經幹細胞的增生、新生神經的分化都有顯著提升。此外，我們發現AAV-β-catenin不只能夠感染神經前驅細胞並造成自泌作用，也能夠感染星狀膠質細胞及中間神經元，而這可能會導致對成體神經新生活動的旁泌作用。由於Wnt/β-catenin的活化曾經被報導過可能在阿茲海默症中具有保護神經元的潛力，且在阿茲海默症疾病模式APP J20小鼠中有退化的神經新生現象，我們利用APP J20阿茲海默症疾病模式小鼠作為探討AAV-β-catenin在阿茲海默症神經新生中所扮演角色的模型。我們首先檢查了實驗室所培育的APP J20小鼠其神經新生的情形，然而只發現輕微的Sox2+及DCX+細胞減少。也因此，顱內注射AAV-β-catenin並沒有辦法在此品系之APP J20小鼠中達成促進神經新生或其他影響的效果。
關鍵字
BCAS2 ； β-catenin ； 成體神經新生 ； BCAS2條件式基因剔除小鼠(CAMKIIα-Cre ； BCAS2flox/flox) ； 阿茲海默症 ； APP J20阿茲海默症疾病模式小鼠並列摘要
Breast Carcinoma Amplified Sequence 2 (BCAS2) is a 26 kDa nuclear protein and a negative regulator of tumor suppressor p53. BCAS2 participates in cell growth regulation and it is a core membrane of Prp19/CDC5L spliceosome complex, therefore essential for pre-mRNA splicing. Previous studies in our lab have shown that BCAS2 regulate β-catenin pre-mRNA splicing, thence β-catenin protein level reduces when BCAS2 depletion in CaMKIIα driven BCAS2 cKO mice. Moreover, lack of BCAS2 interrupts dendrite development in newborn neurons in DG of hippocampus as well as displays impaired learning and memory. We previously find reduced percentage of proliferating NSCs in lenti-shBCAS2 intracranial injected WT mice and the proliferation of NSCs and β-catenin level were rescued after injecting AAV-BCAS2 into DG of BCAS2 cKO mice. The role of β-catenin in adult neurogenesis has been well documented. Wnt/β-catenin signaling promotes self-renewal of neuron stem cells as well as induces differentiation, we hypothesize that BCAS2 regulates NSCs proliferation via β-catenin. In this study, we determine whether the growth of DCX+ newborn neurons would also be affected by BCAS2. Data show impaired number of DCX+ neurons in both BCAS2 cKO mice and lentivirus mediated BCAS2 knockdown mice, while DCX+ neurons restore after intracranial injecting AAV-BCAS2 into DG of BCAS2 cKO mice. Results indicate that BCAS2 regulates neuron differentiation, too. To verify the hypothesis that BCAS2 affects adult neurogenesis directly through β-catenin, we examine whether forced expression of β-catenin could improve adult neurogenesis in BCAS2 cKO mice. Consequently, we generate AAV9-β-catenin and apply it to BCAS2 cKO mice. Results show improving NSCs proliferation and newborn neuron differentiation detected by stem cell marker Sox2, DNA synthesis marker BrdU, immature neuron marker DCX and proliferating marker Ki67. Besides, we find that AAV9-β-catenin not only infect into neuron progenitor cells, which represent an autonomous effect, but also infect astrocytes and interneurons, which may induce paracrine effect on neurogenesis. Additionally, due to the potential benefit of neuron protection by activating Wnt/β-catenin in Alzheimer’s disease and the decreasing neurogenesis in APP mice as previous reports, we use APP J20 mice as a disease model to explore the role of AAV-β-catenin in neurogenesis of AD. We determine the situation of adult neurogenesis in elder APP mice firstly and find only a mild impairment in Sox2+ cell number and DCX+ cell number. For this reason, intracranial injection of AAV-β-catenin cannot make improvement or difference on APP J20 mice line breeding by our lab.