YAP與Rab18調節成年哺乳動物腦中側腦室下區之神經幹細胞功能

Abstract

成年神經幹細胞存在於哺乳動物大腦中的兩個位置，一個位於側腦室的腦室下區（subventricular zone, SVZ），另一個位於海馬齒狀回的顆粒下區（subgranular zone, SGZ）。在胚胎發育過程中，YAP和Sonic hedgehog（SHH）訊息傳遞路徑會維持神經幹細胞並抑制其分化。實驗室先前研究發現YAP會激活SHH訊息傳遞路徑來抑制胚胎皮質前驅細胞中的神經元分化，此外YAP在成年SVZ中會維持神經幹細胞功能。故我們假設YAP也會通過SHH訊息傳遞路徑調節成年神經幹細胞的行為。我們先檢查了成年大腦中兩個SHH訊息傳遞路徑的蛋白質PTCH1和Gli2之表達，發現它們存在於SVZ中。我們以Cre-loxP系統專一剔除小鼠SVZ神經幹細胞中的yap並觀察PTCH1和Gli2之蛋白質表達，發現Gli2表現量下調，表示YAP確實會調控SHH訊息傳遞路徑。另一方面我們在神經球 (neurosphere)培養實驗中SHH訊息傳遞路徑是否介導YAP對神經幹細胞功能的影響。我們發現Gli1的下降抑制了YAP誘導的神經球形成，表示SHH訊息傳遞路徑在YAP的下游並在發育階段和成年階段調節神經幹細胞。Rab18是屬於Ras相關的小GTP酶Rab家族之成員，它調節神經內分泌細胞系中多巴胺的釋放。實驗室先前研究發現Rab18以non-autonomous的方式調節成年神經元新生，通過抑制多巴胺分泌來增加腦中催乳素濃度來增進成年神經元新生。在此我們探討Rab18是否也能透過autonomous作用影響出生後神經元新生。我們取出生後第7天小鼠SVZ培養的初代神經球之神經幹細胞，用針對Rab18的兩種shRNA來降低Rab18表達，發現Rab18不影響神經幹細胞分化成神經元或星狀膠質細胞，但它是神經幹細胞增殖所必需的。另一方面我們發現Rab18的下降使第二代神經球形成有減少的趨勢，表示Rab18有可能會維持神經幹細胞自我更新。綜合以上發現，YAP和Rab18會調節出生後SVZ中的成年神經元新生。

Neural stem cells (NSCs) exist in two places in the adult mammalian brain, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. During embryonic development, YAP and Sonic hedgehog (SHH) pathway maintain NSCs and inhibit their differentiation. Previously, we find that YAP activates the SHH pathway to inhibit neuronal differentiation in the embryonic cortical progenitor cells. In addition, YAP maintains NSCs in the adult SVZ. We hypothesize that YAP also regulates the behavior of adult NSCs through the SHH pathway. First, we examined the expression pattern of PTCH1 and Gli2, two SHH pathway proteins in the adult brain and found that they were present in the SVZ. We studied whether their expression was downregulated by SVZ-specific knockout of yap with Cre-loxP system. We found that knockout of YAP decreased Gli2 expression. Our finding suggests that YAP regulates the expression of Gli2. Furthermore, we studied whether the SHH pathway mediated the effect of YAP on postnatal NSC functions with the neurosphere culture. We found that knockdown of Gli1 inhibited YAP-induced neurosphere formation. Our finding suggests that the SHH pathway acts downstream of YAP to regulate the properties of NSCs both in the developmental and adult stage. In addition, Rab18, a member of Ras-related small GTPase superfamily, regulates Ca2+-mediated exocytosis of dopamine in neuroendocrine cell lines. Previous, we find that Rab18 regulates adult neurogenesis in a cell non-autonomous manner that it increases the prolactin level in the brain by inhibiting dopamine secretion. Here, we examined whether Rab18 also controlled postnatal neurogenesis autonomously. We decreased Rab18 expression with two shRNA constructs against Rab18 in NSCs derived from primary neurospheres of postnatal day seven SVZ and found that Rab18 did not affect the differentiation of NSCs into neurons or astrocytes. Interestingly, it was required for proliferation of NSCs. Furthermore, we found that knockdown of Rab18 may inhibit neurosphere formation. Our finding suggests that Rab18 may regulate the properties of NSCs in the postnatal SVZ. Taken together, our results suggest that YAP and Rab18 regulate adult neurogenesis in the postnatal SVZ.