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標題: | BCAS2 在調控成體神經新生過程之角色 The role of BCAS2 in regulating adult neurogenesis |
作者: | Shih-Hao Lin 林詩浩 |
指導教授: | 陳小梨(Show-Li Chen) |
關鍵字: | BCAS2,成體神經新生,β-catenin,神經幹細胞, BCAS2,adult neurogenesis,β-catenin,neural stem cells(NSCs), |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | BCAS2是一個26kD的核蛋白,身為腫瘤抑制因子p53的負調控者且能夠調控細胞生長。此外,BCAS2 是Prp19/CDC5L 剪切複合體的核心一員,透過調控
Delta pre-mRNA剪切去影響果蠅的生存。Wnt/β-catenin信號通路的功能失調涉及幾種神經退化性疾病,目前我們已經透過微陣列分析知道BCAS2的RNA表現量在阿茲罕默症的病人中有所下降但不了解具體的原因。先前我們使用Cre/LoxP系統生成由主要在海馬迴和皮質中表現的iCAMKIIα所驅動的前腦特異性條件式BCAS2基因剔除小鼠,牠展現了類似小頭畸形症的症狀,樹突神經生長缺陷和學習記憶受損的情況,且有部分原因是因為β-catenin蛋白的表現量下降,顯示出BCAS2能夠調控神經的生長。先前我們未發表的實驗結果顯示條件式BCAS2基因剔除小鼠的神經幹細胞數量比較少(Sox2)增生率也比較低,表示其成體神經新生受損。然而在Sox2表現的神經幹細胞中的iCAMKIIα的表現只有相當低的3.3%。為了避免Sox2細胞中低表現的iCaMKII導致結果難以解釋,我們選擇進行顱內注射能夠敲落BCAS2基因的慢病毒以進一步證實BCAS2在調控神經幹細胞自我更新和增生所扮演的角色。在這項研究中,我們進行立體定向顱內注射攜帶shBCAS2的慢病毒以敲落正常小鼠海馬迴中的內生型BCAS2,並利用腹腔注射溴化去氧尿苷再進行免疫螢光分析去觀察SGZ中的Sox2細胞數量以及增生。實驗結果顯示在海馬迴內BCAS2被敲落小鼠的SGZ中神經幹細胞的數量和增生有顯著的降低,而那些BCAS2被敲落的細胞中β-catenin的表現量也跟著下降,此外,在整個海馬迴當中的β-catenin的蛋白表現量也是下降的。綜合前面的實驗結果來看,在正常小鼠的海馬迴中敲落BCAS2基因可以部分通過β-catenin去減少神經幹細胞的數量及增生。為了進一步研究對條件式BCAS2基因剔除小鼠進行BCAS2基因治療是否可以挽救受損的成體神經新生,我們製造了帶有BCAS2基因的重組腺相關病毒作為治療工具(AAV-CMV-BCAS2)。實驗結果證明AAV-CMV-BCAS2可在接受治療小鼠的海馬迴中表現且AAV基因治療可以成功挽救條件式BCAS2基因剔除小鼠中減少的神經幹細胞數量及增生率。總體而言,我們證實了BCAS2透過β-catenin調控神經幹細胞的自我更新和增生並調節海馬迴的成體神經新生。 Breast carcinoma amplified sequence 2 (BCAS2) is a 26kD nuclear protein. BCAS2 is a negative regulator of tumor suppressor p53 and takes part in cell growth regulation. Besides, BCAS2 is a core component of Prp19/CDC5L spliceosome complex and is essential for Drosophila viability by regulating Delta pre-mRNA splicing. Dysfunctional of Wnt/β-catenin signaling pathway is involved in several neurodegenerative diseases. Currently BCAS2 RNA is reportedly declined in the microarray of analysis of Alzheimer’s disease (AD) patients but not characterize it. Previously, we used Cre/LoxP system to generate forebrain-specific BCAS2 conditional knockout (cKO) mice driven by iCAMKIIα, which expresses predominantly in hippocampus and cortex. BCAS2 cKO mice exhibit microcephaly-like, dendritic malformation, impaired learning and memory phenotypes partially results from the down-regulation of β-catenin; indicating BCAS2 regulates neuron growth. Previously, our unpublished results have demonstrated that BCAS2 cKO mice displayed low neuron stem cell (NSC) numbers (Sox2-positive) and reduced proliferation rate of Sox2, indicating the impairment of adult neurogenesis. However, CAMKIIα-Cre expression in Sox2-expressing NSCs was considerably low about 3.3%. To avoid low iCaMKIIα-Cre expression in Sox2 cells, that cause difficult interpretation for results, we alternatively performed lenti-shBCAS2 intracranial hippocampal injection to further confirm BCAS2-regulating NSC self-renewal and proliferation. In this study, we performed stereotaxic intracranial hippocampal injection of lentivirus carrying shBCAS2 to knockdown BCAS2 in hippocampus of wild type (WT) mice. We administrated BrdU and performed immunofluorescence analysis to examine Sox2 cell number and Sox2 proliferation in SGZ (subgranular zone). The results showed a significant decrease of NSC number and proliferation in SGZ of hippocampal BCAS2-knockdown mice; and low expression level of β-catenin in hippocampus was along with BCAS2-knockdown cells. Furthermore, the β-catenin protein expression from hippocampus lysates was declined in BCAS2-knockdown mice. In sum, specific knockdown BCAS2 in hippocampus can reduce NSCs number and proliferation that is at least partially via β-catenin. In addition, to investigate whether BCAS2 gene therapy on BCAS2 cKO mice could rescue impaired adult neurogenesis, we generated recombinant adeno-associated virus (AAV) carrying BCAS2 as a therapeutic tool to performed BCAS2 gene delivery in BCAS2 cKO mice. Results indicated that AAV gene therapy could rescue the reduced number and proliferation of NSC in cKO mice; and western blot analysis also showed that AAV-CMV-BCAS2 could be expressed in hippocampus of AAV-CMV-BCAS2 injected mice. Taken together, BCAS2 regulates NSC self-renewal and proliferation through a β-catenin-dependent manner. Here, we provide the solid evidences to confirm the role of BCAS2 in regulating adult hippocampal neurogenesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70580 |
DOI: | 10.6342/NTU201802969 |
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顯示於系所單位: | 微生物學科所 |
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