Rab18調控出生後小鼠側腦室下區神經幹細胞之自我更新及成年母鼠的氣味辨識
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2020
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成年哺乳動物的腦中，成年神經幹細胞存在於側腦室的腦室下區（subventricular zone, SVZ）。在SVZ中的神經幹細胞會產生神經母細胞，其沿著rostral migratory stream (RMS) 爬行到嗅球，分化成成熟神經元。在嗅球中新生的神經元主要是負責氣味辨識以及母性行為。Rab18是Rab蛋白的一員，屬於Ras相關的小GTP水解酶家族。過去研究中我們發現Rab18對於維持神經幹細胞增殖是必要的，進而得知Rab18對於成年神經元新生的重要性，但並不影響神經幹細胞分化成神經元或星狀膠質細胞。為了確認這點，我們在神經幹細胞中過表達Rab18以觀察其對神經幹細胞增殖的影響，取出生後第7天小鼠SVZ之神經幹細胞培養成初代神經球後，把神經球打散後送入Rab18全長之質體進行觀察，發現過表達Rab18後促進神經幹細胞的分裂。同時取Rab18基因剔除小鼠的神經幹細胞培養成初代神經球，統計神經球的大小及數量，結果顯示剔除Rab18後，其神經球的體積較小，但不影響其數量。由此上述結果得知Rab18對於神經幹細胞的分裂是必要且充分的。另外，神經幹細胞還有一個標誌性的特徵:自我更新的能力。為了測試Rab18對於神經幹細胞自我更新和分裂的影響，我們取得Rab18基因剔除小鼠SVZ之神經幹細胞培養成初代神經球後打散，將其培養成第二代神經球，統計神經球的大小及數量，結果顯示剔除Rab18後第二代神經球的體積較小，數量上亦顯著較少。由此上述結果得知Rab18對於神經幹細胞自我更新的能力是必要的。然而Rab18如何調控神經幹細胞分裂的機制尚未明確，因此我們以Rab18的shRNA來降低Rab18表達，以及將Rab18全長之質體送入P19細胞中，觀察Rab18表現量的改變對於與細胞週期相關之Cyclin D1的影響，證實Rab18對於Cyclin D1的蛋白質表現是充分且必要。為了了解Rab18如何調控神經幹細胞增殖，我們以螢光素酶報告基因檢測了Rab18與三條訊息傳遞路徑:包括Wnt，Notch和Shh訊息傳遞路徑是否有交互作用。發現Rab18會活化Wnt，Notch和Shh路徑。過去在動物模型中發現Rab18基因剔除小鼠的嗅覺有缺陷，不能分辨其幼鼠的氣味，所以我們進一步檢驗該基因剔除小鼠在一般氣味辨識上是否同樣的缺陷。在研究中使用了化學性氣味進行氣味辨識之行為實驗，發現Rab18對於小鼠記住氣味的能力是必要的。綜合以上發現可知Rab18對神經幹細胞増殖必需且充分的，對於神經幹細胞自我更改亦是必要的，並作用於Wnt，Notch和Shh路徑上游，以及與小鼠維持嗅覺記憶的能力相關。
In the mammalian brain, adult neural stem cells (NSCs) exist in the subventricular zone (SVZ) of the lateral ventricle. In the SVZ, NSCs produce neuroblasts and they migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB) and differentiate into mature neurons. Functions of adult OB neurogenesis are odor discrimination and maternal behaviors. Rab18 is a member of Rab proteins, which belong to Ras-related superfamily of small GTPase. Previously, we find that Rab18 is required for adult neurogenesis by regulating NSC proliferation, but not differentiation. We hypothesize that Rab18 is also sufficient for NSC proliferation. To test it, we overexpressed Rab18 in NSCs derived from postnatal day seven SVZ. We found that overexpression of Rab18 increased cell proliferation. In addition, we cultured NSCs from the SVZ of Rab18-/- mice to form 1’ neurospheres (NSs). In the Rab18-/- group, we found that there was a decrease in the sphere size but no difference of the number of 1’ NSs. These results show that Rab18 is necessary and sufficient for NSC proliferation. NSCs has another feature, self-renewal. We hypothesize that Rab18 is also required for NSC self-renewal and proliferation. To test it, we cultured NSCs from the SVZ of Rab18-/- mice to form 2’ NSs. In the Rab18-/- group, we found that there was a decrease in the sphere size and the number of 2’ NSs. These results show that Rab18 is necessary for NSC self-renewal. To study the mechanism how Rab18 regulates NSCs proliferation, we decreased Rab18 expression with two shRNA constructs against Rab18 or overexpression Rab18 to observe the expression of the cell cycle related protein Cyclin D1. We found that Cyclin D1 protein expression was decreased when Rab18 was knocked down and overexpression Rab18 increased Cyclin D1 protein expression. Our finding suggests that Rab18 is necessary and sufficient for Cyclin D1 protein expression. To find out the downstream signaling pathway of Rab18, we focused on three pathways: Wnt, Notch and Shh signaling pathway. After overexpression of Rab18, we found that the activity of Wnt, Notch and Shh was increased. This result show that Rab18 is the upstream for the Wnt, Notch and Shh pathway. Previously, we find that female Rab18-/- mice have odor discrimination defects that they cannot discriminate odors of their own pups from foreign ones. We further examined whether they had deficits in chemical odor discrimination. We found that Rab18-/- mice had impaired odor memory in chemicals as well. Our finding suggests that Rab18 is required for odor memory. Taken together, our studies suggest that Rab18 regulates NSC proliferation, self-renewal and odor memory in mice, also Rab18 is the upstream for the Wnt, Notch and Shh pathway.
In the mammalian brain, adult neural stem cells (NSCs) exist in the subventricular zone (SVZ) of the lateral ventricle. In the SVZ, NSCs produce neuroblasts and they migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB) and differentiate into mature neurons. Functions of adult OB neurogenesis are odor discrimination and maternal behaviors. Rab18 is a member of Rab proteins, which belong to Ras-related superfamily of small GTPase. Previously, we find that Rab18 is required for adult neurogenesis by regulating NSC proliferation, but not differentiation. We hypothesize that Rab18 is also sufficient for NSC proliferation. To test it, we overexpressed Rab18 in NSCs derived from postnatal day seven SVZ. We found that overexpression of Rab18 increased cell proliferation. In addition, we cultured NSCs from the SVZ of Rab18-/- mice to form 1’ neurospheres (NSs). In the Rab18-/- group, we found that there was a decrease in the sphere size but no difference of the number of 1’ NSs. These results show that Rab18 is necessary and sufficient for NSC proliferation. NSCs has another feature, self-renewal. We hypothesize that Rab18 is also required for NSC self-renewal and proliferation. To test it, we cultured NSCs from the SVZ of Rab18-/- mice to form 2’ NSs. In the Rab18-/- group, we found that there was a decrease in the sphere size and the number of 2’ NSs. These results show that Rab18 is necessary for NSC self-renewal. To study the mechanism how Rab18 regulates NSCs proliferation, we decreased Rab18 expression with two shRNA constructs against Rab18 or overexpression Rab18 to observe the expression of the cell cycle related protein Cyclin D1. We found that Cyclin D1 protein expression was decreased when Rab18 was knocked down and overexpression Rab18 increased Cyclin D1 protein expression. Our finding suggests that Rab18 is necessary and sufficient for Cyclin D1 protein expression. To find out the downstream signaling pathway of Rab18, we focused on three pathways: Wnt, Notch and Shh signaling pathway. After overexpression of Rab18, we found that the activity of Wnt, Notch and Shh was increased. This result show that Rab18 is the upstream for the Wnt, Notch and Shh pathway. Previously, we find that female Rab18-/- mice have odor discrimination defects that they cannot discriminate odors of their own pups from foreign ones. We further examined whether they had deficits in chemical odor discrimination. We found that Rab18-/- mice had impaired odor memory in chemicals as well. Our finding suggests that Rab18 is required for odor memory. Taken together, our studies suggest that Rab18 regulates NSC proliferation, self-renewal and odor memory in mice, also Rab18 is the upstream for the Wnt, Notch and Shh pathway.
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Keywords
Rab18, 神經幹細胞, 化學性氣味辨識, Rab18, Neural stem cells, Chemical odor discrimination