- 學位論文
BCAS2可以調節PSD95並改善在APP轉基因小鼠中所看到類似阿茲海默症的表現型
BCAS2 regulates PSD95 and ameliorates the AD-like phenotype seen in APP transgenic mouse
摘要
BCAS2 (Breast carcinoma amplified sequence 2) 是一個26kD的核蛋白且為Prp19/CDC5L complex中的一個核心蛋白質。BCAS2可以調節精原細胞的剪接並維持早期胚胎發育的基因組完整性。它同時也是p53的負調控者並在DNA受到損傷時扮演一個重要的角色。除此之外，它也是個和雄性激素受器有交互作用且對於前列腺癌細胞的生長與存活是不可或缺的；另外在乳癌病患中，低表現量的BCAS2和較高的無復發生存率有關聯。我們實驗室近期的研究發現BCAS2可以調節β-catenin的RNA剪切。透過Cre/loxP的系統在小鼠出生後，專一性的剃除前腦中的BCAS2基因，而這隻轉基因小鼠有著類似腦小症的表現型，無論在前腦的重量上有下降或是透過組織外觀來觀察都得以證實。再者，和WT小鼠相比之下，條件式BCAS2基因剃除鼠有著較短的樹突長度並從學習和記憶的行為學上發現有著認知上的缺失。在一項關於阿茲海默症病患的微陣列研究結果發現BCAS2的下降並認為其屬於阿茲海默症相關的基因。阿茲海默症是一個最為常見的失智症類型也是一種漸進式的神經退化性疾病，它有著許多神經病理學的改變，舉例而言突觸喪失就是其中一個。發炎反應則在許多神經退化性疾病中都有觀察到，一項近期的研究顯示補體因子例如C1q和C3會協同小膠質細胞作用造成早期阿茲海默症疾病鼠的突觸喪失。PSD95是位於突觸後的一個蛋白質，在阿茲海默症疾病鼠中觀察到因為突觸喪失所造成的PSD95量下降。 在我們近期尚未發表的研究結果顯示BCAS2可以結合到PSD95的mRNA上並且調節其選擇性剪接和表現量。我的研究中發現了條件式BCAS2基因剃除鼠在海馬迴中DG的molecular layer有顯著性的PSD95表現下降以及和突觸前的一個蛋白質Synapsin I的共同定位亦有下降的現象。造成這部分的結果是由於先前實驗室學姊發現在同樣這區域有著小膠質細胞增生的現象， BCAS2又是一個p53的負調節者，而p53和小膠質細胞增生又是有關聯的。因此，上述內容更加強化了BCAS2和PSD95之間的關聯。 因此，我們假設BCAS2可能在具有神經保護的效果且其在阿茲海默症中所扮演的角色亦是我們有興趣的。首先，我們測定了5個月大的J20 APP轉基因小鼠中的BCAS2和PSD95蛋白質表現量，並且發現在海馬迴中有著顯著的下降，皮質區則沒有這個趨勢。接著我們生產了帶有BCAS2基因的慢病毒(lentivirus)質體，透過腦立體定位手術將此病毒打入三個半月小鼠的海馬迴的DG區域。一個半月之後，小鼠則會進行三種行為學實驗來評估BCAS2基因治療的效果。和控制組的APP轉基因小鼠相比之下，有進行BCAS2基因治療的APP轉基因老鼠，其在行為學passive avoidance中的記憶力有著顯著的增加，而另外兩項行為學rotarod和Morris water maze則沒有顯著的差異。最後，我們測量了C1q、PSD95以及兩者的共定位表現量來研究BCAS2基因治療可能的機制。我們發現APP/BCAS2小鼠在特定區域中有著PSD95表現量的上升，而這可能是因為會表現BCAS2的慢病毒所造成的效果。總而言之，BCAS2可以調節PSD95並改善在APP轉基因小鼠中所看到類似阿茲海默症的表現型。
並列摘要
Breast carcinoma amplified sequence 2 (BCAS2) is 26 kDa nuclear protein and a member of the core proteins in the Prp19/CDC5L complex. BCAS2 can regulate the splicing in spermatogonia and maintain genome integrity of early embryonic development. It is also a negative regulator of p53 tumor suppressor and plays a role in response to DNA damage. Aside from that, BCAS2 is an androgen receptor (AR)-interacting protein which is necessary for the survival and growth of prostate cancer cells and lower levels of BCAS2 is associated with higher relapse-free survival of breast cancer patients. Our recent study discovered that BCAS2 can regulate β-catenin RNA splicing. By using the Cre/loxP system, we generated CaMKIIα-iCre drived BCAS2 conditional knockout mice. The transgenic mice have a microcephaly-like phenotype, as evidenced by a decreased of forebrain weight and histological appearance. Besides that, shorter dendrite length is found when compared with WT mice and also display cognitive deficits in learning and memory behavior tests. A human Alzheimer's disease (AD) microarray study showed a down-regulation of BCAS2 and described as an AD-corrlelated genes. Alzheimer's disease, the most common type of dementia and progressive neurodegenerative disease, has many neuropathological alterations, synapse loss for instance. Inflammation has been seen in many neurodegenerative diseases. A recent study showed complement factors such as C1q and C3 along with microglia will mediate early synapse loss in Alzheimer mouse models. Being a marker at the postsynaptic density, PSD95 is lowered in AD mice due to the loss of synapses. Recently our unpublished results discovered BCAS2 could bind with PSD95 mRNA and regulate its alternative splicing and expression. In my study, we found a significant lower post- (PSD95) and co-localized synaptic density (with pre-synaptic marker Synapsin I) in the hippocampus dentate gyrus molecular layer in BCAS2 cKO mice. This part of the data can be a result toward the discovery of microgliosis in this region since BCAS2 can negatively regulate p53 expression which is associated with microgliosis. This strengthens the relationship between BCAS2 and PSD95. Therefore, we hypothesized that BCAS2 might function for neuroprotection and the role of BCAS2 in AD would be interesting for investigation. Firstly, both BCAS2 and PSD95 protein levels were checked in 5-month-old J20 APP mice and found a significant decreased in the hippocampus region but not cortex. We then generated a lentiviral plasmid containing BCAS2 gene. By performing brain stereotaxic surgery, lentivirus was injected into hippocampus dentate gyrus at the age of 3.5-month-old. 1.5 month after virus injection, mice were tested in three behavior tests to evaluate the effect of BCAS2 gene therapy. BCAS2 treatment in APP mice showed a significant improvement in passive avoidance comparing with APP/GFP mice; but not rotarod performance and the Morris water maze test. The levels of C1q, PSD95 and their co-localization were further measured to investigate the possible mechanism of BCAS2 gene therapy. We found a region-specific increase of PSD95 level in the dentate gyrus of APP/BCAS2 mice, which is possibly the effect of the lentiviruses. Collectively, BCAS2 may regulate PSD95 and ameliorate the AD-like phenotype seen in APP transgenic mouse.