Rab18在嗅球成年神經元新生中的作用及分子機制

Abstract

成年神經幹細胞存在於哺乳動物大腦側腦室的腦室下區並且終其一生會負責成年神經元新生。成年神經幹細胞會產生神經母細胞，這些細胞會藉由rostral migratory stream遷徙到嗅球分化成神經元。嗅球成年神經元新生的功能為氣味辨別和育幼行為。Rab18屬於Ras相關的小GTP酶家族之成員。我們發現出生後的腦室下區中，Rab18維持神經幹細胞的增殖。Wnt / -catenin訊號會促進在腦室下區中的細胞增殖。Wnt路徑的其中一個下游基因為Cyclin D1，此蛋白可以增加發育中或是成年大腦中神經幹細胞的數量。我們發現Rab18能夠活化Wnt路徑以及對於Cyclin D1的表現是充分且必要的。因此我們假設Rab18透過-catenin來調控Cyclin D1的表現。我們在P19細胞株中過量表現Rab18，發現Rab18會增加-catenin的表現與促進-catenin進入細胞核。但在P19細胞株中過量表現Rab18並且降低-catenin表現，發現Cyclin D1的表現依舊增加，顯示Rab18不是透過-catenin來調控Cyclin D1的表現。銀杏內酯A (GA)是銀杏萃取物的其中一種成分。我們發現GA能夠活化Wnt路徑，並且促進Cyclin D1與 -catenin的表現。因此我們假設減少Rab18的表現時，加入GA能挽救Cyclin D1與 -catenin的表現。在P19細胞株中降低Rab18表現並且加入GA，發現Cyclin D1與 -catenin的表現量增加，顯示GA能挽救Rab18缺失時，Cyclin D1與 -catenin的表現。我們也發現Rab18能活化Sonic hedgehog (Shh) 路徑。Shh路徑的下游轉錄因子Gli2會調控Cyclin D1基因的表現。所以我們假設 Rab18 透過 Gli2 調節Cyclin D1的表達。在P19細胞株中過量表現Rab18並且降低Gli2表現，發現Gli2缺失會反轉Rab18促進Cyclin D1的效果。在過往研究中，我們發現Rab18-/-產後母鼠對於費洛蒙的氣味辨別有缺陷，牠們無法區分自己幼崽和外來幼崽的氣味。Rab18-/-處女母鼠對於一般氣味的記憶也是受損的。所以我們想進一步檢查Rab18-/-處女鼠和產後母鼠中，一般氣味辨別和氣味偵測是否存在缺陷。我們發現 Rab18 對於處女和產後母鼠的氣味辨別是必要的。Rab18對氣味偵測也是必須的。我們的研究顯示Rab18促進-catenin的表達以及使其進入到細胞核並透過 Gli2而非-catenin來促進Cyclin D1的表現。而GA能挽救因為Rab18缺失的Cyclin D1與 -catenin表現。此外Rab18對於處女鼠和產後小鼠能夠維持一般氣味的相關行為是必須的。

Adult neural stem cells (NSCs) exist in the subventricular zone (SVZ) of the lateral ventricle in the mammalian brain and are responsible for neurogenesis throughout life. In the SVZ, NSCs produce neuroblasts, which migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB) and differentiate into neurons. Functions of adult OB neurogenesis are odor discrimination and maternal behaviors. Rab18 is a member of the Ras-related small GTPase superfamily. Previously, we find that Rab18 maintains the proliferation of NSCs in the postnatal SVZ. The Wnt/-catenin signaling has been shown to promote cell proliferation in the SVZ. One of the Wnt pathway target genes is Cyclin D1, which promotes the expansion of NSC in the developing and adult brain. We find that Rab18 activates the Wnt pathway and is necessary and sufficient for the expression of Cyclin D1. Therefore, we hypothesized that Rab18 regulated the expression of Cyclin D1 through-catenin. First, we overexpressed Rab18 in P19 cells. We found that Rab18 promoted the expression of -catenin and its translocation into the nucleus. Second, we overexpressed Rab18 and knocked down -catenin in P19 cells. We found that Rab18 did not promote the expression of Cyclin D1 through -catenin. Ginkgolide A (GA) is one of the components of Ginkgo biloba extract. We find that GA activates the Wnt pathway and promotes the expression of Cyclin D1 and -catenin. Therefore, we hypothesized that GA could rescue Cyclin D1 and -catenin expression in Rab18 knockdown group. We knocked down Rab18 and treated cells with GA. We found that GA rescued Cyclin D1 and -catenin expression in Rab18 knockdown group. We discover that Rab18 also activates the Sonic hedgehog (Shh) pathway. Gli2 has been shown to mediate the mitogenic effects of Shh by transcriptional activation of cyclin D1. Therefore, we hypothesized that Rab18 regulated the expression of Cyclin D1 throughGli2. We overexpressed Rab18 and knocked down Gli2 in P19 cells. We found that Rab18 promoted the expression of Cyclin D1 through Gli2. Previously, we find that female Rab18-/- mice have odor discrimination defects that they cannot discriminate odors of their own pups from foreign ones. Rab18-/- female virgin mice have impaired odor memory in chemicals as well. We further examined whether they had deficits in chemical odor discrimination and odor detection in female virgin and postpartum mice. We found that Rab18 was necessary for odor discrimination in female virgin and postpartum mice. Rab18 was also required for odor detection in female virgin and postpartum mice. Taken together, our studies suggest that Rab18 promotes -catenin expression and its translocation into the nucleus, and Rab18 promotes the expression of Cyclin D1 through Gli2. GA rescues Cyclin D1 and -catenin expression in Rab18 loss-of-function condition. In addition, Rab18 is required for odor behaviors in female virgin and postpartum mice.