鋰鹽改善BCAS2剔除小鼠的神經缺損

Abstract

BCAS2是一個26 kD的核蛋白，為p53的負調控者進而影響細胞生長。此外，BCAS2透過增加雄激素受體mRNA的表現量和維持蛋白質的穩定性來促進前列腺癌細胞細胞增生。BCAS2參與形成Prp19/CDC5L complex，調控活化核醣核酸的剪切。BCAS2透過調控Delta pre-mRNA剪切去影響果蠅生存和翅膀發育。先前我們在外顯子微陣列分析指出β-catenin是BCAS2的目標基因，也證明 BCAS2是β-catenin剪切的正調控者。Wnt/β-catenin訊息傳遞路徑曾被報導在許多神經退化性疾病中受到阻礙。此外，近期文獻指出在微陣列分析中，阿茲海默症病患的BCAS2 RNA表現量下降；在涵蓋BCAS2的基因座位中的基因變異也和自閉症相關，暗示BCAS2參與神經新生。我們實驗室利用Cre/loxP系統在小鼠出生後，專一性剔除前腦BCAS2基因，造成前腦體積變小、樹突發育異常及學習和記憶能力下降，推測可能原因為β-catenin表現量下降。 在本篇研究中，我們探討是否可以透過提高β-catenin表現量來改善BCAS2條件式基因剔除小鼠的神經退化現象。鋰鹽已知透過抑制GSK-3β來活化Wnt/β-catenin訊息傳遞路徑，刺激神經細胞增生，故我們欲了解鋰鹽是否可以成為改善BCAS2條件式基因剔除小鼠的神經退化現象的治療物。首先，我們探討鋰鹽對細胞的影響，在分化的N2A細胞默化BCAS2，發現細胞的絲狀偽足(Filopodia)數目和β-catenin表現有減少現象。並在默化BCAS2同時外送β-catenin大量表現，發現絲狀偽足數目減少現象有明顯的回復。接著，探討鋰鹽是否可以改善BCAS2條件式基因剔除小鼠神經退化現象。經由小鼠前腦切片染色，發現在小鼠海馬迴中CA1的stratum radiatum和DG的 molecular layer有微膠細胞增生的現象但沒有星狀膠細胞增生現象，此結果暗示在小鼠前腦條件式基因剔除BCAS2會造成神經細胞的缺損。此外，也發現BCAS2條件式基因剔除小鼠有前腦體積變小，學習和記憶力衰退的現象。在進行行為實驗前，我們以open field test 和 visible platform tests確認條件式基因剔除小鼠的活動力和視力正常。經由小鼠行為實驗發現鋰鹽透過活化Wnt/β-catenin訊息傳遞路徑來提高β-catenin表現量，改善BCAS2條件式基因剔除小鼠的學習和記憶能力以及減緩DG的 molecular layer微膠細胞增生的現象。此結果暗示BCAS2透過調控β-catenin去影響小鼠學習和記憶能力。目前已有諸多文獻報導，Wnt/β-catenin訊息傳遞路徑會調控成體神經新生(adult neurogenesis)。在成體神經新生時，神經幹細胞會增生或分化成成熟的神經細胞，去執行學習或記憶。透過小鼠前腦切片染色發現條件式剔除BCAS2會造成神經幹細胞(SOX2)數目減少，並且锂鹽可以有效恢復條件式剔除小鼠的神經幹細胞數目。接著，以溴脫氧尿核苷(BrdU)標定新生細胞的結果也顯示，條件式基因剔除小鼠減少的增殖神經幹細胞(proliferating neural stem cells)可以透過锂鹽刺激而增加。故實驗結果顯示，BCAS2可以調控神經幹細胞的增殖和活化。近來有文獻報導指出CaMKII在神經幹細胞(Nestin)的表現量極少(約2%)，而我們的BCAS2條件式剔除小鼠同樣式是透過CaMKII去驅動進行。透過小鼠前腦切片染色發現我們WT小鼠的CaMKII-Cre在神經幹細胞(SOX2)的表現量約3.3%。欲精確探討BCAS2對神經幹細胞或前驅細胞的影響，經由小鼠前腦切片以及Cre，SOX2和BrdU染色結果，更加確認我們的條件式基因剔除小鼠成功經由Cre/loxP系統剔除BCAS2基因進而導致神經幹細胞的增殖和活化能力下降。以上結果暗示鋰鹽透過活化Wnt/β-catenin訊息傳遞路徑來提高β-catenin表現量，進而改善BCAS2條件式基因剔除小鼠的學習和記憶能力並提高增殖神經幹細胞的數目。

Breast carcinoma amplified sequence 2 (BCAS2) is a 26 kD nuclear protein, a negative regulator of p53 and participates in cell growth regulation; and promotes prostate cancer cells proliferation by enhancing androgen receptor (AR) mRNA transcription and protein stability. BCAS2 is involved in Prp19/CDC5L spliceosome complex for regulation of RNA splicing and is essential for Drosophila viability and wing development by controlling Delta pre-mRNA splicing. Exon array analysis indicated that β-catenin was one of BCAS2-targeted gene. We currently proved that BCAS2 is up-regulator for β-catenin splicing. Disruption of Wnt/β-catenin signaling is involved in several neurodegenerative diseases such as Alzheimer’s disease (AD). Current report of other group demonstrated that the RNA expression of BCAS2 is reduced in the microarray analysis of AD patients. Also, genetic variant on BCAS2 gene linkage region (1p13.2) is associated with the risk of autism, indicating BCAS2 may be associate with neural related disorder. Recently, we used the Cre/loxP system driven by CaMKIIα to generate BCAS2 conditional knock out (cKO) in postnatal forebrain, which causes microcephaly-like, dendritic malformation, poor learning and memory phenotypes partly reasons from down-regulation of β-catenin. In this study, we would investigate whether elevated β-catenin could rescue BCAS2 cKO causing neuron degeneration. Lithium, an inhibitor of glycogen synthase kinase-3β (GSK-3β), leads to the activation of the Wnt/β-catenin signaling pathway and an increase in β-catenin. Therefore, we were interested to investigate whether lithium can be a therapeutic agent for the depletion of BCAS2-causing neural defect. Firstly, we measured the effect of lithium in cells; knockdown of BCAS2 in differentiated N2A cell caused defect in filopodia formation along with the reduction of β-catenin. The overexpression of β-catenin could rescue impaired filopodia formation in silenced BCAS2 expression N2A cells. In sum, lithium treatment could increase β-catenin level along with increased numbers of filopodia in BCAS2 knockdown-differentiated N2A cells. Next, we examined whether lithium could rescue BCAS2 cKO causing neural degeneration. The reactive gliosis in cKO mice was measured and compared with WT mice. The selective microgliosis was found in the cornu ammonis1 stratum radiatum (CA1 Rad) and dentate gyrus molecular layer (DG Mo) but did not exhibit astrogliosis in cKO compared with WT mice, which confirmed depletion of BCAS2 in the forebrain caused neural defect. Additionally, BCAS2 cKO mice have small brains with impaired learning and memory abilities. The open field test and visible platform tests were conducted to ensure the mobility and visual acuity of cKO mice were comparable as WT. And lithium treatment to cKO mice could improve their spatial learning and memory capability using passive avoidance and Morris water maze; and reduced selective microgliosis in the molecular layer (DG Mo) compared with untreated cKO mice. Additionally, lithium treatment could increase β-catenin expression in cortex and hippocampus both in WT and cKO mice. These results further support that BCAS2 regulates cognitive learning and memory through β-catenin. Wnt/β-catenin signaling reportedly regulates the adult hippocampal neurogenesis. During neurogenesis, neuron stem cells (NSCs) can proliferate to maintain stem cell pool or differentiate to be mature granule cells for learning and memory storage. Therefore, we used IFA analysis to compare Sox2 expression cell between WT and cKO mice and found lower Sox2-positvie NSCs in cKO than WT mice. The declined number of Sox2-positvie NSCs in cKO mice which were restored by lithium treatment. Moreover, we administrated BrdU and IFA analysis to further examine the role of BCAS2 in growth of newborn neurons. We found that the declined number of Sox2 co-expressed with BrdU cells in cKO mice which were recovered by lithium treatment. Taken together, BCAS2 can regulate neuron stem cell activation/proliferation. Current report demonstrated that CaMKII expression rarely occurs in Nestin-expressing cells (about 2%). BCAS2 cKO mice were driven by CaMKII-Cre; the proportion of Sox2 stem cell in Cre transgenic WT mice was examined about 3.3% by measuring the percentage of Cre+Sox2+ cells to Sox2 single-positive cells in the SGZ. Hence to further precisely characterize BCAS2 effect on neuron stem/progenitor cells, we measured the percentage of Cre+Sox2+ cells to Cre single-positive cells in the SGZ in WT (CaMKIIα-Cre; BCAS2+/+) and BCAS2 cKO (CaMKIIα-Cre; BCAS2Flox/Flox). The results revealed that a higher percentage of Cre+Sox2+ cells to Cre single-positive cells in the SGZ in the lithium-treated group than in PBS-treated group both in WT and cKO mice. Furthermore, after BrdU administration, we evaluated the percentage of Cre+Sox2+BrdU+ cells to Cre single-positive cells in the SGZ, similar results displayed that BCAS2 plays a role in the maintenance of NSCs proliferation in SGZ and lithium administration increases NSCs proliferation both in the SGZ of WT and cKO mice. Collectively, lithium treatment can improve cognitive learning and memory and rescue Sox2 stem cell activation/proliferation in BCAS2 cKO mice.